Developing accurate, stable, facile, and highly sensitive detection methods is crucial for monitoring low levels of benzoylurea insecticides (BUs) which are priority pollutants harmful to ecological environment and human health. Herein, Fe3O4@OH-CMPs with triazine and -OH functionalized groups and large specific surface area (351.9 m²·g-¹), were synthesized covalently at room-temperature and utilized in the magnetic solid-phase extraction process of BUs. DFT calculation and characterization analyses demonstrated that BUs were bonded to Fe3O4@OH-CMPs primarily via π-π stacking and O-H···F hydrogen bond. Quantitative approach revealed lower limits of detection (0.19 to 1.2 ng·L-¹), higher reusability, and was applicable to more complicated sample matrices in comparison with other methods. Furthermore, the successful determination of BUs emitted from water, fruit juices, and tea samples with recovery (71.7-112.4 %) indicated the wide applicability of Fe3O4@OH-CMPs. This work accomplished a swift and sensitive detection of BUs, highlighting the potential of Fe3O4@OH-CMPs in maintaining food and environmental safety.

Abstract Background: Snake venom C-type lectin-like proteins (also known as snaclecs) have anticoagulation and procoagulation effects by targeting platelet or coagulation factor IX/X, suggesting their potential as candidates for new anticoagulant drugs. Therefore, this study aims to evaluate the antiplatelet and antithrombotic effects of a new snaclec from Protobothrops mucrosquamatus venom and its potential as an anticoagulant candidate. Methods: Promucetin was purified through sequential column chromatography, and its molecular mass was determined by SDS-PAGE. The α- and β-chains of promucetin were identified using liquid chromatography-mass spectrometry (LC-MS). In vitro analyses of platelet aggregation were performed using turbidimetric methods, thromboelastography, and coagulation activity assays. For in vivo experiments, promucetin was administered to rats at varying concentrations, and platelet changes were monitored. The antithrombotic effects of promucetin were assessed using a FeCl₃-induced rat thrombosis model. Results: Promucetin existed as two multimers with molecular weights of 140.1 kDa and 91.9 kDa under non-reducing conditions. Sequence analysis revealed that its α-chain and β-chain shared 71% and 34% homology, respectively, with TMVA from the same snake venom. In vitro platelet aggregation assays indicated that promucetin activated platelets via glycoprotein Ib. Thromboelastography showed that promucetin inhibited both coagulation factor activity and platelet function, resulting in an anticoagulant effect. Specifically, thrombin time was prolonged, while activated partial thromboplastin time and prothrombin time remained unchanged. In vivo, promucetin administration led to a dose-dependent decrease in platelet count. At doses of 25 and 50 μg/kg, promucetin significantly inhibited thrombosis, with inhibition rates of 40.9% and 74.4%, respectively. For comparison, lysine acetylsalicylate produced an inhibition rate of 36.7%. Conclusion: Promucetin exhibits significant ability to modulate coagulation function and effectively inhibit thrombosis by activating platelet via GPIb and reducing platelet count, which helps us understand its biological function in snake bites, it exhibits the potential to be a candidate for anticoagulant therapy.

Acute lung injury (ALI) is an acute respiratory-related progressive disorder, which lacks specific pharmacotherapy. Icariin (ICA) has been shown to be effective in treating ALI. However, the targets and pharmacological mechanisms underlying the effects of ICA in the treatment of ALI are relatively lacking. Based on network pharmacology and molecular docking analyses, the gene functions and potential target pathways of ICA in the treatment of ALI were determined. In addition, the underlying mechanisms of ICA were verified by immunohistochemistry, immunofluorescence, quantitative Real-time PCR, and Western blot in LPS-induced ALI mice. The biological processes targeted by ICA in the treatment of ALI included the pathological changes, inflammatory response, and cell signal transduction. Network pharmacology, molecular docking, and in vivo experimental results revealed that ICA inhibited the complement C5a-C5aR1 axis, TLR4 mediated NF-κB, MAPK, and JAK2-STAT3 signaling pathways related gene and protein expressions, and decreased inflammatory cytokine, chemokine, adhesion molecule expressions, and mitochondrial apoptosis in LPS-induced ALI.

Snake venoms, comprising a complex array of protein-rich components, an important part of which are snake venom metalloproteinases (SVMPs). These SVMPs, which are predominantly isolated from viperid venoms, are integral to the pathology of snakebites. However, SVMPs derived from elapid venoms have not been extensively explored, and only a handful of SVMPs have been characterized to date. Atrase A, a nonhemorrhagic P-III class metalloproteinase from Naja atra venom, exhibits weak proteolytic activity against fibrinogen in vitro but has pronounced anticoagulant effects in vivo. This contrast spurred investigations into its anticoagulant mechanisms. Research findings indicate that atrase A notably extends the activated partial thromboplastin time, diminishes fibrinogen levels, and impedes platelet aggregation. The anticoagulant action of atrase A primarily involves inhibiting coagulation factor VIII and activating the endogenous fibrinolytic system, which in turn lowers fibrinogen levels. Additionally, its effect on platelet aggregation further contributes to its anticoagulant profile. This study unveils a novel anticoagulant mechanism of atrase A, significantly enriching the understanding of the roles of cobra venom metalloproteinases in snake venom. Furthermore, these findings underscore the potential of atrase A as a novel anticoagulant drug, offering insights into the functional evolutions of cobra venom metalloproteinases.

Snake venom C-type lectin-like proteins (CLPs) belong to the nonenzymatic proteins. To date, no CLP with both platelet and coagulation factors activating activities has been reported. In this study, a novel CLP, termed protocetin, with molecular weight of 29.986 kDa, was purified from the Protobothrops mucrosquamatus venom (PMV). It consists of alpha- and beta-chains, with 67% similarity in their N-terminal sequence. Protocetin activates glycoprotein Ib (GPIb) by binding to von Willebrand factor (vWF), inducing platelet aggregation. It also activates the intrinsic coagulation pathway by binding to coagulation factor IX. After injection of protocetin into mice at dose of 0.5 mu g/g or 1.5 mu g/g, it resulted in activation of platelets, a notable reduction in platelet count and prolonged tail bleeding time. Additionally, the plasma activated partial thromboplastin time (APTT) was significantly extended, and the fibrinogen concentration was markedly reduced. Thrombelastogram comfirmed the anticoagulation effect of protocetin. Notably, no microthrombosis was observed in tissues of lung, liver and kidney within 1 h after injection of protocetin into the mice at dose of 0.5 mu g/g. This study revealed protocetin as a novel CLP from PMV that has dual functions in activating platelet and coagulation factor IX, thereby modulates coagulation in vivo. This work contributes to a better understanding of the structure and function of snake venom CLP.

Acute lung injury (ALI) as one kind of acute pulmonary inflammatory disorder, manifests primarily as damage to alveolar epithelial cells and microvascular endothelial cells. Activation of the complement system is a common pathological mechanism in ALI induced by diverse factors, with the complement alternative pathway assuming a pivotal role. Baicalin, a flavonoid derived from the root of Scutellaria baicalensis Georgi, exhibits noteworthy biological activities. The present study attempted the interventional effects and underlying mechanisms of baicalin in microangiopathy in ALI induced by complement alternative pathway activation. Activation of the complement alternative pathway by cobra venom factor (CVF). HMEC cells were pretreated with baicalin and then exposed to complement activation products. The expression of inflammatory mediators was detected by ELISA, and the intranuclear transcriptional activity of NF-κB was assessed by a dual fluorescent kinase reporter gene assay kit. Before establishing the ALI mouse model, baicalin or PDTC was gavaged for 7 d. CVF was injected into the tail vein to establish the ALI model. The levels of inflammatory mediators in BALF and serum were determined by ELISA. HE staining and immunohistochemistry evaluated pathological changes, complement activation product deposition, and NF-κB p65 phosphorylation in lung tissue. Baicalin reduced complement alternative activation product-induced expression of HMEC cells adhesion molecules (ICAM-1, VCAM-1, E-selectin) and cytokines (IL-6, TNF-α) as well as upregulation of NF-κB intranuclear transcriptional activity. Baicalin intervention reduced the number of inflammatory cells and protein content in the BALF and decreased the levels of IL-6, TNF-α, and ICAM-1 in serum and IL-6, TNF-α, ICAM-1, and P-selectin in BLAF. In addition, baicalin attenuated inflammatory cell infiltration in the lung of ALI mice and reduced the deposition of complement activation products (C5a, C5b-9) and phosphorylation of NF-κB p65 in lung tissue. Baicalin relieves complement alternative pathway activation-induced lung inflammation by inhibition of NF-κB pathway, delaying the progression of ALI.

Endothelial dysfunction and pathological alterations are pivotal in the pathogenesis of cardiovascular disease. To date, effective interventions for these endothelial changes are lacking. Tumor necrosis factor-alpha (TNF-α) is known to significantly contribute to these alterations. It has been reported the potential of luteolin to mitigate TNF-α-induced inflammation, yet its specific mechanisms and targets still remain to be elucidated. This study aims to investigate the effects and mechanisms of luteolin on TNF-α-induced inflammatory injury in human microvascular endothelial cells, thereby advancing the understanding of luteolin’s medicinal properties. Our findings demonstrate that luteolin notably inhibits TNF-α-induced phosphorylation of Akt, mitogen activated protein kinase (MAPK), and the nuclear factor-kappaB (NF-κB) p65. It significantly reduces the transcriptional activity of NF-κB p65 and AP-1 and decreases the expression of mRNA and proteins related to adhesion molecules and inflammatory mediators. Additionally, luteolin inhibited the reduction in STAT3 phosphorylation. In conclusion, luteolin effectively suppresses TNF-α-induced inflammatory injury in endothelial cells via the Akt/MAPK/NF-κB pathway.

Snake venom metalloproteinases (SVMPs), which are a critical component of viperid and crotalid venoms, play various important roles in the pathogenesis of snakebite envenomation. The SVMPs from elapid venoms are not well elucidated, as compared with those from viperid and crotalid venoms. Atrase A is a nonhemorrhagic P-III SVMP purified from Naja atra venom that possesses only weak fibrinogenolytic activity. In our prior study, we found that atrase A detached adherent cells from the substrate. In this work, we investigated further the effect and mechanism of atrase A on endothelial cells. Oxidative damage, inflammatory mediators, apoptosis, and activation of the NF-κB and MAPK signaling pathways were measured after HMEC-1 cells were exposed to atrase A. The results showed that HMEC-1 cells released inflammatory mediators, exihibited oxidative damage and apoptosis after exposure to atrase A. The Western blot analysis results revealed that atrase A increased Bax/Bcl-2 and caspase-3 levels and activated the NF-κB and MAPK signaling pathways in endothelial cells. The effects on endothelial cells were nearly completely abolished after atrase A was treated with ethylenediamine tetraacetic acid. These results showed that atrase A led to an inflammatory response, cellular injury and apoptosis in endothelial cells, and this effect was due to its metalloproteinase domain. The study contributes to a better understanding of the structures and functions of cobra venom P-III class metalloproteinases.

Phytochemical investigation on the bioactive constituents of the tubers of Lepidium meyenii led to two new quinoline derivatives (1 and 2) and one new macamide (5), together with six known compounds (3, 4, and 6–9). The new structures were elucidated by means of the extensive spectroscopic analyses. The NMR data of compound 3 were firstly reported and its structure was affirmed by the X-ray crystallography diffraction. Compound 9 showed moderate AChE inhibitory activity, and 1, 3, and 8 exhibited moderate inhibitory effect on the secretion of TNF-α by RAW264.7 cells.

原矛头蝮蛇(Protobothrops mucrosquamatus)主要分布于我国长江以南和台湾地区,原名烙铁头蛇(Trimeresurus mucrosquamatus),属蝰蛇科原矛头蝮属[1].原矛头蝮蛇毒(Protobothrops mucrosquamatus venom,PMV)主要表现为血液毒性,影响心血管及凝血系统等方面,对组织细胞也有毒性作用[2-3].该蛇伤较为常见,临床表现主要为凝血障碍、头昏恶心、视力模糊和胸闷腹痛等,严重者出现皮下出血、血尿、全身内脏出血甚至失血性休克[4-5].迄今,对PMV的研究主要集中在纤溶酶、类凝血酶、丝氨酸蛋白酶及诱导血小板聚集的蛋白等成分[1-2,6].课题组前期研究表明,PMV具有明显的破坏凝血功能的作用,PMV及其组分能诱导血小板聚集引起全血中血小板计数下降,具有酶切底物S-2251活性,能延长凝血酶时间(TT)和部分活化凝血活酶时间(APTT),破坏人微血管内皮细胞[2,7].此前研究更多采用凝血四项等研究方法,为进一步研究和认识PMV对凝血功能的影响,课题组采用血栓弹力图测定方法来开展PMV对大鼠全血凝血功能影响的研究,以期为进一步认识原矛头蝮蛇伤的机制及临床救治方案和新药研究提供参考.

OBJECTIVE To investigate the toxic effect and mechnism of kaempferol on human kidney proximal tubular epithelial cells （HK-2） based on high content analysis technology.METHODS HK-2cells were divided into the control group （final concentration of dimethylsulfoxide＜0.01%）,kaempferol 510,20,50 and 100μmol·L -1 and doxorubicin (Dox,final concentration 10μmol·L -1 ) groups.The count of living cells was detected via Hoechst 33342 staining,the cell viability was determined by MTT assay,the levels of reactive oxygen species （ROS）,mitochondrial membrane potential （MMP） and the intracellular calcium ion were detected by fluorescence probes of DCFH-DA,Mito-Tracker Red CMXRos and Fluo-4 AM.The ATP content was measured with the chemiluminescence method,and apoptosis by flow cytometry.The DNA content was detected by Hoechst 33342 staining.The nuclear translocation of NF-κB p65,the expressions of p38 mitogen-activated protein kinases （p38 MAPK）,p-p38 MAPK,extracellular signal regulated kinase （ERK）,p-ERK and c-Jun N-terminalkinases （JNK）,p-JNK protein in associated mitogen activated protein kinases （MAPK） signal pathway,and those of signal transducer and activator of transcription-3 （STAT3）,p-STAT3 protein in janus kinase-2 （JAK2）/STAT3 signal signa pathway were detected by immunofluorescence assay.RESULTS Compared with the control group,the count of living cells in the kaempferol 5,10,20,50 and 100μmol·L -1 groups and the cell viability in the kaempferol 10,20,50 and 100μmol·L -1 groups were significantly deceased （P＜0.05,P＜0.01）.The levels of ROS were significantly increased in the kaempferol 10,20,50 and 100μmol·L -1 groups （P＜0.05,P＜0.01）,while those of MMP in 5,10,20,50 and 100μmol·L -1 groups were obviously decreased（P＜0.01）.The levels of calcium influx in kaempferol 50,100μmol·L -1 groups were increased significantly（P＜0.05,P＜0.01）,while the ATP content in the kaempferol 50 and 100μmol·L -1 groups was obviously decreased （P＜0.05,P＜0.01）.The apoptotic rates in kaempferol 20,50,100μmol·L -1 groups were up-regulated significantly （P＜0.05,P＜0.01）,but the DNA content hardly changed in all the kaempferol groups.The results of immunofluorescence assay showed that kaempferol 50 and 100μmol·L -1 led to the phosphorylation and nuclear translocation of NF-κB p65,the expressions of p-p38 MAPK,p-ERK,p-JNK and JNK of MAPK signaling pathway were up-regulated significantly,and that the expression of p-STAT3protein of JAK2/STAT3 signaling pathway was also increased （P＜0.05,P＜0.01）.CONCLUSION Kaempferol exerts toxic effect on HK-2 cells,and its mechanism is probably related to oxidative stress injury and apoptosis mediated by the up-regulation of ROS levels.

Aim To investigate the effect of phillygenin（PHI） on lipopolysacchride（LPS） and normal human plasma（NHP） induced inflammatory injury on alveolar type II epithelial A549 cells and the related mechanism. Methods A549 cells were exposured to 1 mg·L -1 of LPS and 5% NHP to build the inflammatory injury model. The effects of PHI on cell viability, cell number, area, morphology, and DNA content of A549 cells were detected by thiazole blue colorimetric（MTT） and Hoechst 33342 staining. The A549 cells were pretreated with PHI for 2 h, and then exposed to 1 mg·L -1 of LPS and 5% NHP. The contents of inflammatory cytokines and NF-κB p65 were determined by ELISA and quantitative real-time PCR. The transfer of NF-κB p65 from cytoplasm to nucleus was detected by cellular immunofluorescence. The protein expressions of NF-κB and MAPK signaling pathways were detected by Western blot. Results The contents of IL-6 and IL-8 and the transfer of NF-κB p65 from cytoplasm to nucleus were significantly up-regulated in LPS combined with NHP co-stimulated group. PHI at 1, 10, 50, and 100 μmol·L -1 decreased the mRNA and protein expressions of IL-6 and IL-8, inhibited the transfer of NF-κB p65 from cytoplasm to nucleus, reduced the NF-κB p65 mRNA level, down-regulated the phosphorylation levels of IκBα, NF-κB p65, p38, JNK, and ERK, and up-regulated the protein expression of IκBα in a dose-dependent manner. Conclusions LPS combined with NHP successfully induces inflammatory injury in alveolar type II epithelial A549 cells. PHI could improve the inflammatory response by inhibiting the activation of LPS-TLR4-NF-κB/MAPK signaling pathways.

Background: Acteoside, a water-soluble active constituent of diverse valuable medicinal vegetation, has shown strong anti-inflammatory property. However, studies on the anti-inflammatory property of acteoside in complement-induced acute lung injury (ALI) are limited. Therefore, this study aims to evaluate the anti-inflammatory activity of acteoside in cobra venom factor (CVF)-stimulated human microvascular endothelial cells (HMEC) and in ALI mice model. Methods: In this study, we investigated the effects of acteoside (20, 10, and 5 μg/mL) in vitro in CVF induced HMECs and the activity of acteoside (100, 50, and 20 mg/kg/day bodyweight) in vivo in CVF induced ALI mice. Each eight male mice were orally administered acteoside or the positive drug PDTC (100 mg/kg/day) for 7 days before CVF (35 μg/kg) injection. After injection for 1 h, the pharmacological effects of acteoside were investigated by spectrophotometry, pathological examination, enzyme-linked immunosorbent assay, and immunohistochemistry. Results: In vitro, acteoside (20, 10, and 5 μg/mL) reduced the protein expression of adhesion molecules and pro-inflammatory cytokines and transcriptional activity of NF-κB (P < 0.01). In vivo studies showed that acteoside dose-dependently alleviated lung histopathologic lesion, inhibited the production of the protein content of BALF, leukocyte cell number, lung MPO activity, and expression levels of IL-6, TNF-α, and ICAM-1, and suppressed the C5b-9 deposition and NF-κB activation in CVF-induced acute lung inflammation in mice (P < 0.05, 0.01). Conclusion: This study demonstrates that acteoside exerts strong anti-inflammatory activities in the CVF-induced acute lung inflammation model and suggests that acteoside is a potential therapeutic agent for complement-related inflammatory diseases.

Aflatoxin B1 (AFB1), a mycotoxin contaminating food and feed, can trigger liver immune toxicity and threaten the poultry industry. Phillygenin (PHI) is a natural lignan derived primarily from Forsythia suspensa with hepatoprotective pharmacological and medicinal properties. This research aimed to investigate the preventive effects of PHI on the toxicity of AFB1 in the liver of chickens. Chickens were administered with AFB1 (2.8 mg/kg) and/or treated with PHI (24 mg/kg) for 33 days. The histopathological changes, serum biochemical indices, oxidative damage, inflammatory mediators, apoptosis, and activation of the NF-κB and Nrf2 signaling pathways were measured. Results revealed that dietary PHI ameliorated liver function indicators, reduced the malondialdehyde and inflammatory mediator production and the apoptotic cell number, and increased the antioxidant enzyme contents and Bcl-2 level. The quantitative realtime PCR and Western blot results revealed that PHI reduced p53, cytochrome c, Bax, caspase-9, and caspase-3 levels, normalized the NF-κB p65 phosphorylation, and upregulated the Nrf2 and its downstream genes expression in chicken liver. These results indicated that PHI has beneficial effects on AFB1-induced liver damage, oxidative damage, inflammatory response, apoptosis, and immunotoxicity by inhibiting NF-κB and activating the Nrf2 signaling pathway in chickens. This study provides new insight into the therapeutic uses of PHI.

目的 研究中华眼镜蛇毒对大鼠凝血功能的影响.方法 体外实验采用中华眼镜蛇毒及其分离峰与大鼠全血孵育,测定血栓弹力图;体内采用尾静脉或肌肉注射眼镜蛇毒,取不同时间点全血测定血栓弹力图;取体内外实验分离的血浆测定LDH活力、血红蛋白、vWF、P-selectin含量并采用酶切发色底物法测定凝血相关酶活性.结果 体内外实验结果表明,眼镜蛇毒及其部分分离峰可明显引起大鼠凝血功能的异常,表现为凝血因子减少、纤维蛋白原水平低下、血小板功能减弱;同时大鼠血浆血红蛋白含量及LDH活力显著升高.体外实验中眼镜蛇毒及其部分分离峰可显著下调酶切凝血酶及纤溶酶底物活性.体内实验中肌肉注射后血浆中P-selectin含量0.5h时显著升高,酶切凝血因子Ⅹa底物活性6h内显著上调;尾静脉注射后血浆酶切凝血酶底物活性2h内显著上调,6 h内vWF含量明显升高.结论 中华眼镜蛇毒能明显引起大鼠凝血功能的异常,其作用主要与激活凝血系统导致凝血因子消耗、纤维蛋白原减少以及血小板消耗等有关.

Introduction Disseminated intravascular coagulation (DIC) is a syndrome characterized by coagulopathy, microthrombus, and multiple organ failure. The complement system in DIC is overactivated, and the functions of complement and coagulation pathways are closely related. Our previous screening revealed that salvianolic acid A (SAA) has anti-complement activity. The hyper-activated complement system was involved in the lipopolysaccharide (LPS) induced DIC in rats. The effects of SAA anti-complement action on LPS-induced DIC in rats were investigated. Methods The complement activity of the classical pathway and alternative pathway was detected through an in vitro hemolysis assay. The binding sites of SAA and complement C3b were predicted by molecular docking. LPS-induced disseminated coagulation experiments were performed on male Wistar rats to assess coagulation function, complement activity, inflammation, biochemistry, blood routine, fibrinolysis, and survival. Results SAA had an anti-complement activity in vivo and in vitro and inhibited the complement activation in the classical and alternative pathway of complement. The infusion of LPS into the rats impaired the coagulation function, increased the plasma inflammatory cytokine level, complemented activation, reduced the clotting factor levels, fibrinogen, and platelets, damaged renal, liver, and lung functions, and led to a high mortality rate (85%). SAA treatment of rats inhibited complement activation and attenuated the significant increase in D-dimer, interleukin-6, alanine aminotransferase, and creatinine. It ameliorated the decrease in plasma levels of fibrinogen and platelets and reversed the decline in activity of protein C and antithrombin III. The treatment reduced kidney, liver, and lung damage, and significantly improved the survival rate of rats (46.2 and 78.6% for the low- and high-dose groups, respectively). Conclusion SAA reduced LPS-induced DIC by inhibiting complement activation. It has considerable potential in DIC treatment.

Forsythia suspensa (Thunb.) Vahl (Oleaceae) leaves are valuable sources of phillygenin. This study aimed to isolate phillygenin from F. suspensa leaves and examine its analgesic and anti-inflammatory effects. Phillygenin was successfully extracted and isolated from F. suspensa leaves after fermentation. Phillygenin significantly reduced the number of writhing induced by acetic acid, prolonged the latency period in the hot plate test, and inhibited the xylene-induced ear edema and carrageenan-induced paw edema in mice. IL-6, TNF-α, IL-1β, NO, and PGE2 levels in the carrageenan-induced paw edema were notably reduced after pretreatment with phillygenin. Phillygenin significantly decreased the iNOS and COX-2 protein expressions and the IκB-α and NF-κB p65 phosphorylation. This study demonstrated that phillygenin is a potential therapeutic candidate for managing pain and inflammation-mediated disorders. The study contributes to the comprehensive development and utilization of F. suspensa leaves for economic and health care. Practical applications Phillygenin is one of the major active ingredients in Forsythia suspensa. But the content of phillygenin in F. suspensa is very low which limits its application. Phillygenin has potential pharmacological activity and anti-inflammatory properties. However, the potential effects of phillygenin on analgesic activity have not been clarified. Furthermore, the data on its anti-inflammatory activity in vivo are relatively limited. This study evaluated the analgesic activity for the first time and the acute anti-inflammatory effect of phillygenin from F. suspensa leaves by fermentation, which indicated phillygenin is a potential therapeutic candidate for managing pain and inflammation-mediated disorders.

目的 利用高内涵筛选技术研究吴茱萸次碱(rute-acarpine,RUT)的肝毒性及其可能机制.方法 HepG2细胞暴露于不同浓度的RUT后作用不同时间,MTT法检测细胞活力,采用高内涵检测RUT对细胞存活、细胞核面积、线粒体膜电位(MMP)、ROS、钙离子内流、细胞膜完整性(DIR)和MAPK、NF-κB、JAKs-STATs信号通路活化水平的影响,流式细胞检测细胞凋亡.结果 100 μmol·L-1 RUT明显抑制了HepG2细胞的活力(P＜0.01),表现为RUT作用24 h后,细胞核面积减少,核形态不均一,作用48 h后存活细胞数量明显减少(P＜0.01),并伴随着细胞的早期凋亡(P＜0.01);从6 h开始,100 μmol·L-1 RUT组细胞内活性氧和钙离子水平明显升高(P＜0.01),细胞膜完整性明显降低(P＜0.01);100 μmol·L-1 RUT 作用 24 h 后,ERK1/2、JNK、STAT3 和p38的磷酸化水平明显升高(P＜0.01,P＜0.05),总蛋白未见明显变化;在3 h可检测到c-Jun、c-Fos的表达上调,与对照组相比,差异具有显著性(P＜0.01);在3 h时间点,可明显检测到p-NF-κB p65的表达上调(P＜0.01),但NF-κB p65入核不明显.结论 吴茱萸次碱在100 μmol·L-1的条件下对HepG2细胞表现出细胞毒性,其毒性机制主要与氧化应激和炎症反应导致的细胞损伤有关.

A series of novel conjugates of benzoselenazole or selenazole and CPI-1 were designed, synthesized, and evaluated for inhibitory activities against the botulinum neurotoxin A (BoNT/A) light chain (LC) and BoNT/A in vivo. The results show that these compounds exhibit potent inhibitory activities to the LC with IC50 of 0.5-4.1 mu M. The reaction kinetics and the mass spectra of the reaction products of LC with benzoselenazole- or selenazolecoupled CPI-1 demonstrate that the benzoselenazole group of most inhibitors is coupled to the LC of BoNT/A. These data indicate that the CPI-1 conjugates can inhibit both the active center of BoNT/A LC as well as Cys165, therefore functioning as irreversible bifunctional inhibitors. The detoxification activities in vivo show that one of the benzoselenazole-CPI-1 compounds prolongs the survival time of mice challenged by 2 x LD50 of BoNT/A. This work provides a new strategy to design potent antidotes of BoNT/A.