Protective Effects and Potential Mechanism of Tongxinluo on Mice with Thromboangiitis Obliterans Induced by Sodium Laurate.

OBJECTIVE: To investigate the effects of Tongxinluo (TXL) on thromboangiitis obliterans (TAO) and the underlying mechanisms. METHODS: Ninety male C57/BL6J mice were randomly divided into 6 groups according to a random number table: the sham group, TAO model group, Compound Danshen Tablet (CDT) group, and the high-, medium-, and low-dose TXL groups. All mice except the sham group were injected with sodium laurate (0.1 mL, 5 mg/mL) in the femoral artery to establish TAO mouse model. After modeling, mice in the sham and TAO model groups were intragastrically administered 0.5% (w/v) sodium carboxymethylcellulose, mice in the CDT group were intragastrically administered 0.52 g/kg CDT, and mice in the TXL-H, TXL-M, and TXL-L groups were intragastrically administered 1.5, 0.75, and 0.38 g/kg TXL, respectively. After 4 weeks of gavage, the recovery of blood flow in the lower limbs of mice was detected by Laser Doppler Imaging. The pathological changes and thrombosis of the femoral artery were observed by morphological examination. The expressions of tumor necrosis factor α (TNF-α) and inducible nitric oxide synthase (iNOS) in the femoral artery wall were detected by HE staining. Levels of thromboxane B2 (TXB2), 6-keto-prostaglandin F1α (6-keto-PGF1α), endothelin-1 (ET-1), interleukin (IL)-1ß and IL-6 were measured using enzyme-linked immunosorbent assay (ELISA). Levels of activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FIB) were detected by a fully automated biochemical analyzer. RESULTS: TXL promoted the restoration of blood flow in the lower limbs, reduced the area of thrombosis in the femoral artery, and alleviated the pathological changes in the femoral artery wall. Moreover, the levels of TXB2, ET-1, IL-6, IL-1ß, TNF-α and iNOS were significantly lower in the TXL groups compared with the model group (P<0.05 or P<0.01), while the level of 6-keto-PGF1α was significantly higher (P<0.01). In addition, APTT, PT, and TT were significantly prolonged in TXL groups compared with the model group (P<0.05 or P<0.01), and FIB levels were significantly decreased compared with the model group (P<0.01). CONCLUSIONS: TXL had a protective effect on TAO mice, and the mechanism may involve inhibition of thrombosis and inflammatory responses. TXL may be a potential drug for the treatment of TAO.

Integrated pharmacokinetics and pharmacometabolomics to reveal the synergistic mechanism of a multicomponent Chinese patent medicine, Mailuo Shutong pills against thromboangiitis obliterans.

BACKGROUND: Mailuo Shutong Pills (MLST) have displayed pharmacological activity against thromboangiitis obliterans (TAO). However, the active ingredients and therapeutic mechanism of MLST against TAO remained to be further clarified. PURPOSE: The aim of this study was to explore the active components of MLST and their synergistic mechanism against TAO by integrating pharmacokinetics (PK) and pharmacometabolomics (PM). METHODS: TAO model rats were established by sodium laurate solution. Firstly, the efficacy of MLST was evaluated by gangrene score, blood flow velocity, and hematoxylin-eosin (H&E) staining. Secondly, PK research was conducted on bioavailable components to characterize their dynamic behaviors under TAO. Thirdly, multiple plasma and urine metabolic biomarkers for sodium laurate-induced TAO rats were found by untargeted metabolomics, and then variations in TAO-altered metabolites following MLST treatment were analyzed utilizing multivariate and bioinformatic analysis. Additionally, metabolic pathway analysis was performed using MetaboAnalyst. Finally, the dynamic link between absorbed MLST-compounds and TAO-associated endogenous metabolites was established by correlation analysis. RESULTS: MLST significantly alleviated gangrene symptoms by improving the infiltration of inflammatory cells and blood supply in TAO rats. Significant differences in metabolic profiles were found in 17 differential metabolites in plasma and 24 in urine between Sham and TAO rats. The 10 bioavailable MLST-compounds, such as chlorogenic acid and paeoniflorin, showed positive or negative correlations with various TAO-altered metabolites related to glutamate metabolism, histidine metabolism, arachidonic acid metabolism and so on. CONCLUSION: This study originally investigated the dynamic interaction between MLST and the biosystem, providing unique insight for disclosing the active components of MLST and their synergistic mechanisms against TAO, which also shed light on new therapeutic targets for TAO and treatment.

Protective Effects and Potential Mechanism of Tongxinluo on Mice with Thromboangiitis Obliterans Induced by Sodium Laurate / 中国结合医学杂志

OBJECTIVE@#To investigate the effects of Tongxinluo (TXL) on thromboangiitis obliterans (TAO) and the underlying mechanisms.@*METHODS@#Ninety male C57/BL6J mice were randomly divided into 6 groups according to a random number table: the sham group, TAO model group, Compound Danshen Tablet (CDT) group, and the high-, medium-, and low-dose TXL groups. All mice except the sham group were injected with sodium laurate (0.1 mL, 5 mg/mL) in the femoral artery to establish TAO mouse model. After modeling, mice in the sham and TAO model groups were intragastrically administered 0.5% (w/v) sodium carboxymethylcellulose, mice in the CDT group were intragastrically administered 0.52 g/kg CDT, and mice in the TXL-H, TXL-M, and TXL-L groups were intragastrically administered 1.5, 0.75, and 0.38 g/kg TXL, respectively. After 4 weeks of gavage, the recovery of blood flow in the lower limbs of mice was detected by Laser Doppler Imaging. The pathological changes and thrombosis of the femoral artery were observed by morphological examination. The expressions of tumor necrosis factor α (TNF-α) and inducible nitric oxide synthase (iNOS) in the femoral artery wall were detected by HE staining. Levels of thromboxane B2 (TXB2), 6-keto-prostaglandin F1α (6-keto-PGF1α), endothelin-1 (ET-1), interleukin (IL)-1β and IL-6 were measured using enzyme-linked immunosorbent assay (ELISA). Levels of activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FIB) were detected by a fully automated biochemical analyzer.@*RESULTS@#TXL promoted the restoration of blood flow in the lower limbs, reduced the area of thrombosis in the femoral artery, and alleviated the pathological changes in the femoral artery wall. Moreover, the levels of TXB2, ET-1, IL-6, IL-1β, TNF-α and iNOS were significantly lower in the TXL groups compared with the model group (P<0.05 or P<0.01), while the level of 6-keto-PGF1α was significantly higher (P<0.01). In addition, APTT, PT, and TT were significantly prolonged in TXL groups compared with the model group (P<0.05 or P<0.01), and FIB levels were significantly decreased compared with the model group (P<0.01).@*CONCLUSIONS@#TXL had a protective effect on TAO mice, and the mechanism may involve inhibition of thrombosis and inflammatory responses. TXL may be a potential drug for the treatment of TAO.

Protective effects and potential mechanism of salvianolic acid B on sodium laurate-induced thromboangiitis obliterans in rats.

BACKGROUND: The root of Salvia miltiorrhiza f. alba (RSMA) (Lamiaceae) is used for the treatment of patients with thromboangiitis obliterans (TAO) in traditional Chinese medicine. Previously, a mixture of phenolic acids extracted from RSMA has shown significant protective effects on TAO rats. PURPOSE: This study investigates the inhibitory effects of salvianolic acid B on TAO induced by sodium laurate injection in rats to explore the effective constituents of RSMA in TAO treatment. METHODS: TAO rats were developed using injected sodium laurate. After treatment with ligustrazine hydrochloride (15 mg/kg) and various doses of salvianolic acid B (10, 20, 40 mg/kg) by tail intravenous injection, levels of thromboxane B2 (TXB2), 6-keto-prostaglandin F1α (6-keto-PGF1α) and endothelin-1 (ET-1) in plasma were determined using enzyme-linked immunosorbent assay. The right femoral arteries were studied by hematoxylin and eosin staining and immunohistochemical analysis to determine pathological changes and overexpression of tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) in the femoral artery walls of TAO rats. RESULTS: Salvianolic acid B significantly decreased the expressions of TXB2 and ET-1 and increased the expression of 6-keto-PGF1α in plasma, and significantly inhibited the overexpression of TNF-α and iNOS in the femoral artery walls of TAO rats at medium and high doses. CONCLUSION: Salvianolic acid B has a protective effect on TAO rats. The mechanism may involve inhibition of thrombosis and TAO-associated inflammatory responses, which may explain the success of RSMA treatment of TAO in humans in traditional Chinese medical practice. Hence, it may be a potential drug for TAO treatment in conventional medicine.

Effect of bradykinin on rats with thromboangiitis obliterans through PI3K/Akt signaling pathway.

OBJECTIVE: To explore the effect of bradykinin on rats with thromboangiitis obliterans (TAO) through the phosphatidylinositol 3-hydroxy kinase/protein kinase B (PI3K/Akt) signaling pathway. MATERIALS AND METHODS: The female Wistar rats were injected with lauric acid via the femoral artery to establish the TAO model, and they were randomly divided into control group (healthy rats), model group (TAO rats) and bradykinin group (TAO rats injected with bradykinin B2 receptor-specific inhibitor). The control was set in each group before the operation. The level of serum bradykinin in each group was detected via enzyme-linked immunosorbent assay (ELISA), and the reactive oxygen species (ROS) level, Caspase-3 activity and PI3K/Akt protein concentration in vascular tissues were measured via ELISA, Western blotting, ROS assay, and Caspase-3 activity assay, respectively. Moreover, the specific therapeutic mechanism of bradykinin was analyzed. RESULTS: In control group, the intima of the lower extremity venous tissues was smooth, the extima had no evident changes, and there was no inflammatory cell invasion around the arteries and veins. In model group, there was massive inflammatory cell invasion into the lower extremity venous tissues. In bradykinin group, fibrosis and atrophy occurred in venous tissues, the extima was thickened without fibrosis, and there was phagocytosis of neutrophils and mononuclear macrophages around the arteries and veins, as well as massive inflammatory infiltration. The PI3K/Akt protein concentration in lower extremity venous tissues was the highest in control group and the lowest in bradykinin group, and there were statistically significant differences (p<0.01). At 24 h after administration of doxorubicin (DOX), the level of ROS in lower extremity venous tissues was higher in bradykinin group than that in model group (p<0.05), and it was also higher in model group than that in control group (p<0.05). Besides, the activity of Caspase-3 in lower extremity venous tissues was significantly increased in bradykinin group compared with that in model group and control group, while it was slightly higher in model group than that in control group (p<0.05). CONCLUSIONS: The low expression of bradykinin can promote TAO in rats by the mechanism that it inhibits the PI3K/Akt signaling pathway to raise the oxidative stress level, thereby aggravating TAO.

[Marijuana-associated peripheral arteriopathy]. / Arteriopatía periférica asociada a marihuana.

The marijuana arteriopathy should be considered in young patients with peripheral arterial disease with no risk factors for atherosclerosis. It was described for the first time in 1960 and since then there have been about 100 cases published in the literature. Although it tends to be considered as an independent entity of thromboangiitis obliterans or Leo Buerger's disease, in the light of the last findings it is possible to consider it within the spectrum of the latter. We present two cases of young patients with peripheral vascular disease associated with marijuana use where other associated illnesses had been excluded and where the mainstay of treatment has been the cessation of marijuana consumption. It is essential to assess drug use in young patients presenting with peripheral arterial disease.

La arteriopatía por marihuana debe ser considerada en pacientes jóvenes con arteriopatía periférica sin factores de riesgo para ateroesclerosis. Se ha descrito por primera vez en 1960 y existen más de 100 casos en la literatura. Si bien se tiende a considerar como una entidad independiente de la tromboangeítis obliterante o enfermedad de Leo Buerger, debido a los hallazgos la consideramos dentro del espectro de esta última. Presentamos dos casos de pacientes jóvenes con enfermedad vascular periférica asociada al consumo de marihuana, luego de excluir otras enfermedades. El tratamiento es el cese del consumo. Es indispensable valorar el uso de drogas en pacientes jóvenes que se presentan con arteriopatía periférica.

Arteriopatía periférica asociada a marihuana / Marijuana-associated peripheral arteriopathy

La arteriopatía por marihuana debe ser considerada en pacientes jóvenes con arteriopatía periférica sin factores de riesgo para ateroesclerosis. Se ha descrito por primera vez en 1960 y existen más de 100 casos en la literatura. Si bien se tiende a considerar como una entidad independiente de la tromboangeítis obliterante o enfermedad de Leo Buerger, debido a los hallazgos la consideramos dentro del espectro de esta última. Presentamos dos casos de pacientes jóvenes con enfermedad vascular periférica asociada al consumo de marihuana, luego de excluir otras enfermedades. El tratamiento es el cese del consumo. Es indispensable valorar el uso de drogas en pacientes jóvenes que se presentan con arteriopatía periférica.

The marijuana arteriopathy should be considered in young patients with peripheral arterial disease with no risk factors for atherosclerosis. It was described for the first time in 1960 and since then there have been about 100 cases published in the literature. Although it tends to be considered as an independent entity of thromboangiitis obliterans or Leo Buerger´s disease, in the light of the last findings it is possible to consider it within the spectrum of the latter. We present two cases of young patients with peripheral vascular disease associated with marijuana use where other associated illnesses had been excluded and where the mainstay of treatment has been the cessation of marijuana consumption. It is essential to assess drug use in young patients presenting with peripheral arterial disease.

Alleviation of A disintegrin and metalloprotease 10 (ADAM10) on thromboangiitis obliterans involves the HMGB1/RAGE/ NF-κB pathway.

Thromboangiitis obliterans (TAO), also known as Buerger's disease, is a nonatherosclerotic inflammatory disease that influences medium- and small-sized blood vessels of extremities. However, mechanisms underlying TAO are still unclear. As a mediator associated with inflammation, A disintegrin and metalloprotease 10 (ADAM10) was hypothesized to play inhibitory roles in the development of TAO. Thus, the objective of this study is to investigate the effects of ADAM10 in a sodium laurate-induced TAO rat model and elucidate underlying mechanisms. Male Wistar rats were randomly divided into four groups (n = 6) for treatment: sham-operated (SHAM), TAO model (TAO), ADAM10 low dose injection (3 mg/kg; ADAM10-LD) and ADAM10 high dose injection (6 mg/kg; ADAM10-HD). After 14-day treatment, color Doppler ultrasound and hematology analysis indicated TAO rats displayed higher whole blood viscosity and blood platelet count compared with those in the SHAM group. Histologic evaluation and transmission electron microscopy revealed that the ultrastructural damages of vascular smooth muscle and endothelial cells were observed in TAO rats, such as fractured endoplasmic reticulum, decreased cell counts, and fibrillation. On the other hand, the typical signs and symptoms of TAO rats were significantly alleviated via ADAM10 treatment with a dose-dependent pattern. Real-time PCR and western blot results revealed that the expression of high-mobility-group box 1 (HMGB1), receptor for advanced glycation end-products (RAGE) and nuclear factor-kappa B (NF-κB) increased in TAO rats whereas decreased by ADAM10 treatment in both mRNA and protein levels. In conclusion, the results suggest ADAM10 alleviates symptoms of sodium laurate-induced TAO in rats via the RAGE/NF-κB signaling pathway and provides insight into the molecular basis and a potential therapeutic strategy for TAO.

Hypoxia-inducible factor-1α expression in the different stages of rat thromboangiitis obliterans.

We investigated the expression and effects of hypoxia-inducible factor-1α (HIF-1α) in rat thromboangiitis obliterans (TO). Rats were divided into sham and model groups. The model group was further divided into groups based on observation duration. Lauric acid was injected below an artery clamp to simulate TO in the model group; saline was used in the sham group. Clamps were removed 15 min after injection in both groups, and physiological changes were observed at different times (gross observation and hematoxylin and eosin staining). The animals were killed at various times following the operation and serum and muscle tissues were sampled. For the sham group: the endometrium was relatively intact; medial membrane and epineurium lesions were absent; and blood vessels and surrounding tissues had no inflammatory cell infiltration. For the model group: all subgroups displayed inflammation; large numbers of inflammatory cells were gathered; muscle tissue lost its normal texture and structure; and the internal elastic membrane was integrated. Compared with the preoperative status, HIF-1α expression increased significantly in all subgroups (P < 0.05); there was no change in the sham group. HIF-1α expression in each subgroup was different (F = 14.267, P < 0.05). Femoral artery injection of lauric acid can be used as a rat TO model owing to its simple application and success rate. HIF-1α expression increased in the early stage of TO and gradually decreased with the extension of ischemia time; it may play a leading role in TO development and can be used for diagnosis and cure evaluation.

High-mobility-group box protein 1A box reduces development of sodium laurate-induced thromboangiitis obliterans in rats.

OBJECTIVE: High-mobility-group box protein 1 (HMGB1), as a late mediator of inflammation, plays a key role in inflammatory responses by inducing and extending the production of proinflammatory cytokines. The effect of HGMB1 in the inflammatory disease thromboangiitis obliterans (TAO) is unknown. We aimed to investigate the role of HMGB1 in sodium laurate-induced TAO in rats. METHODS: Male Wistar rats were randomly divided into five groups (n=8 each) for treatment: normal, sham-operated, TAO model, and low-dose (15 mg/kg) or high-dose (30 mg/kg) recombinant A box (rA box) infection (administered intraperitoneally once daily for 15 days). The TAO model was induced by sodium laurate and graded by gross appearance on day 15 after femoral artery injection. Histologic changes were measured by histopathology in rat femoral arteries. Plasma levels of HMGB1, thromboxane B2, 6-keto-prostaglandin F1-α, and blood cell counts and blood coagulation levels were measured. Expression of HMGB1, receptor for advanced glycation end-products (RAGE), interleukin-6, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 was assessed by immunohistochemistry and immunofluorescence, Western blot analysis, and quantitative reverse-transcription polymerase chain reaction. RESULTS: The typical signs and symptoms of TAO were observed on day 15 after sodium laurate injection. The expression of HMGB1, RAGE, interleukin-6, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 was markedly increased in rat femoral arteries. Plasma levels of HMGB1 and thromboxane B2 were elevated, but the level of 6-keto-prostaglandin F1-α was decreased. Blood was in a hypercoagulable state, and prothrombin, thrombin, and activated partial thromboplastin times were all significantly shortened, whereas fibrinogen level was increased in TAO rats compared with sham-operated rats. These effects were terminated by the HMGB1 antagonist rA box. CONCLUSIONS: HMGB1 is involved in the inflammatory state in a model of TAO induced by sodium laurate in rats, probably via its receptor RAGE. As the antagonist of HMGB1, rA box can attenuate the development of TAO, which may be a potential therapeutic target for the treatment of TAO.

Urocortin promotes the development of vasculitis in a rat model of thromboangiitis obliterans via corticotrophin-releasing factor type 1 receptors.

BACKGROUND AND PURPOSE: Urocortin is a locally expressed pro-inflammatory peptide. Here we have examined the effects of urocortin on sodium laurate-induced peripheral arterial vasculitis in rats, modelling the mechanisms of thromboangiitis obliterans (TAO). EXPERIMENTAL APPROACH: Peripheral vasculitis in rats was induced by sodium laurate and graded by gross appearance on the 12th day after injection. Histological changes in rat femoral arteries were assessed by histopathology and transmission electron microscopy. Blood cell counts, blood rheology, blood coagulation and plasma urocortin, thromboxane B(2), prostaglandin E(2) and soluble intercellular adhesion molecule-1 levels were measured. Expression of urocortin, corticotrophin-releasing factor (CRF(1/2)) receptors, cyclooxygenase (COX)-2 and intercellular adhesion molecule-1 (ICAM-1) at both mRNA and protein levels were determined by RT-PCR and Western blot. KEY RESULTS: Rats showed grossly visible signs and symptoms of TAO on the 12th day after sodium laurate injection. In these rats, blood was in a hypercoagulable state; plasma urocortin, prostaglandin E(2) and soluble intercellular adhesion molecule-1 levels were elevated; and the expression of urocortin, CRF(1) and CRF(1alpha)-receptors, COX-2 and ICAM-1 in rat femoral arteries were markedly increased. Exogenous urocortin, given for 12 days after sodium laurate, exacerbated the hypercoagulable state and augmented expression of CRF(1alpha)-receptors, COX-2 and ICAM-1. These effects were abolished by a CRF(1)-receptor antagonist, NBI-27914, or a non-selective CRF-receptor antagonist, astressin, but not by the CRF(2)-receptor antagonist, antisauvagine-30, given with exogenous urocortin. CONCLUSION AND IMPLICATIONS: Urocortin exacerbated the hypercoagulable state and vasculitis in a model of TAO induced by sodium laurate in rats, via CRF(1)-receptors. COX-2 and ICAM-1 might also have contributed to this exacerbation.

[Effect of Mailuoning injectable powder on experimental vascular occlusion angeitides in rats].

OBJECTIVE: To investigate the effect of Mailuoning injectable powder on experimental vascular occlusion angeitides in rats. METHODS: Rats were injected laurostearic acid into arteria cruralis to induce the model of experimental vascular occlusion angeitides, then we observed the changes of objective sign of rats, and analysed throm ranking through pathological section under electro-microscope. RESULTS: Mailuoning injectable powder could decrease the quantity of throm in blood vessel, and improve hemorrheoiogy. CONCLUSION: The results show that Mailuoning injectable powder has obvious therapeutical effect on experimental vascular occlusion angeitides in rats, and its mechanism may be related to the anti-throm in blood vessel and improving hemorrheology.

[Cannabis arteritis: four new cases]. / Artériopathies du cannabis: quatre nouveaux cas.

INTRODUCTION: The responsibility of cannabis in juvenile thromboangeitis has been suggested for few years. We describe four new cases. EXEGESIS: Young men presented with distal arteriopathy of the lower limbs in 3 cases, and of the left upper limb in the remaining patient. Symptoms occurred progressively, distal pulses had disappeared, and distal necrosis was constant. Three patients suffered from Raynaud phenomenon, none of them presented with venous thrombosis. Radiologic evaluation revealed distal abnormalities in all cases, and proximal arterial thrombosis in one case. The four patients were cannabis smokers for at least four years. With cannabis interruption and symptomatic treatment, lesions improved for three patients. For one of them, recurrence of arteriopathy occurred when he resumed to smoke cannabis. For the fourth one who never stopped cannabis, an amputation was necessary. CONCLUSION: Search for cannabis use is important because interruption may improve prognosis.

[Review of current etiopathogenic data of Buerger disease]. / Przeglad danych etiopatogenetycznych o chorobie Buergera.

Although 75 years have passed since Buerger's disease was described as a separate nosological, its etiology and pathogenesis are not sufficiently elucidated. According to many authors the disease origin is significantly connected to genetic and environmental factors. Exposure of some patients with special genotype, mainly HLA-A9 and HLA-B5, to environmental factors, mainly nicotine, may be the base of etiology and pathogenesis of Buerger's disease. Discovery of antielastin, anticollagen I and III antibodies, antinicotine and antivascular antigen antibodies in blood of patients, allowed to put forward a theory of immunological character of TO. In Buerger's disease, defined in recent years as an inactive collagenosis, immunological complexes, cell toxins developing during phagocytosis, found in smokers, constitute the main agents responsible for vascular wall damage. Disturbance of prostacyclin I2/thromboxane A2 balance and accelerated platelet aggregation cause spasm of arterioles and in effect lead to higher procoagulant readiness. Some adhesive molecules, for example P and L selectins, play an important role in vascular endothelium damage. Prostaglandin treatment induces an improvement of vascular wall (endothelium) status, and simultaneously improvement of tissue perfusion, expressed by a decrease of selectin and vWF concentrations and of the number of desquamated endothelial cells.