Discovery and development of tricyclic matrinic derivatives as anti-diabetic candidates by AMPKαactivation

We found compound 12N-p-trifluoromethylbenzenesulfonyl matrinane(1)was a potent anti-diabetic agent.Thirty-five tricyclic matrinic derivatives were synthesized and determined for their stimulatory effects on glucose consumption in L6 myotubes,taking 1 as the lead.In high-fat diet(HFD)and STZ induced diabetic mice,9a significantly lowers blood glucose,improves glucose tolerance,and especially alleviates diabetic nephropathy and islet damage.Mechanism study indicates that 9a simultaneously targets mitochondrial complex I to increase AMP/ATP ratio,as well as liver kinase B1(LKB1)and calcium/calmodulindependent protein kinase(Ca MKK),which synergistically activates AMPKαand then stimulates glucose transporter 4(GLUT4)membrane translocation and 2-deoxyglucose(2-DG)uptake to exert anti-diabetic efficacy.Therefore,compound 9a with a novel structure is a promising anti-diabetic candidate with the advantage of multiple-target mechanism,worthy of further investigation.

Synthesis and biological evaluation of 12-benzyl matrinic amide derivatives as a novel family of anti-HCV agents

A new series of 12-benzyl matrinic amide/ethanamide derivatives were synthesized from matrinine（1）and evaluated for their anti-HCV activity,taking compound 2 as the lead.SAR revealed that the introduction of a suitable substituent at the N’-end of matrinic amide might greatly enhance the potency.Among them,matrinic acid 17 and N’-substituted matrinic amides 18a-d exhibited promising potency with low micromolar EC50 values ranging from 1.03μmol/L to 7.54 μmol/L,and better therapeutic window with SI from 66 to 132.Moreover,compound 17 displayed an excellent PK and safety profile in vivo,demonstrating good drug-like characteristics.Thus,it has been selected for further investigation,with an advantage of decreased chances of inducing drug-resistance mutations.

Synthesis and biological evaluation of novel tricyclic matrinic derivatives as potential anti-filovirus agents

Twenty-six novel tricyclic sophoridinic and matrinic derivatives containing a common chlorinated benzene fragment were designed, synthesized and evaluated for their anti-ebolavirus(EBOV)activities. Structure–activity relationship analysis indicated:(i) 12 N-dichlorobenzyl motif was beneficial for the activity;(ii) the chiral configuration at C5 atom might not affect the activity much. Among the target compounds, compound 7d exhibited the most potent potency against EBOV with an IC_(50) value of 5.29 μmol/L and an SI value of over 37.8. Further in vivo anti-EBOV assay of 7d identified its high effectiveness, and in vivo anti-MARV assay of 7d suggested its inspiring broad-spectrum anti-filovirus activity. The results provided powerful information on further strategic optimization and development of this kind of compounds against filoviruses.

ZNF638/NP220同源蛋白及其生物学作用的研究进展

一些具有锌指结构域的蛋白质可以识别特定的靶RNA分子并与之结合,在细胞中参与或介导重要的生物学作用,这一发现拓展了人们对锌指蛋白专职转录调控并且高度依赖于特异结合靶DNA序列的认知。ZNF638/NP220及其同源蛋白Matrin3和RBM20是一类在生物进化中相当保守的特殊锌指蛋白,定位于细胞核基质的核斑中,含有特殊的蛋白结构域,可以特定靶向RNA分子,在细胞的转录调控和RNA加工的不同阶段发挥作用,特别是RNA转录和剪接事件的偶联以及改变RNA分子的成熟和稳定性等方面。本文将对近年来有关ZNF638同源蛋白成员的发现、结构、分子机制及其在细胞分化、胚胎发育、病毒的感染等生命过程中调控作用的研究进展进行总结讨论。

Study on the mechanism of the anti-tumor effects of matrine via regulation of lipid metabolism mediated by SREBP signaling pathway

Objective:To investigate the therapeutic effects of matrine on CT26 tumor-bearing mice and its effect on the sterol regulatory element binding protein(SREBP)signaling pathway.Methods:A CT26 tumor-bearing mouse model was established using CT26 cells.Different doses of matrine were orally administered to mice,and the tumor size and weight in each group of mice before and after administration were measured to calculate the tumor inhibition rate of matrine.Subsequently,tumor tissues were subjected to hematoxylin and eosin(HE)staining to observe morphological changes in tumor tissue,and quantitative polymerase chain reaction(qPCR)was performed to detect the expression of the genes of the lipid metabolism-related enzymes sterol regulatory element binding transcription factor 1(Srebf1),ATP citrate lyase(Acly),acetyl-Coenzyme A carboxylase alpha(Acc),and fatty acid synthase(Fasn)in tumor tissues before and after matrine intervention.Results:Compared to those in the model group,tumor-bearing mice in both the low and high-dose matrine groups showed significantly reduced tumor weights.HE staining showed that matrine significantly inhibited tumor cell proliferation in both the low and high-dose groups.The qPCR results showed that,compared with the model group,the expression levels of the genes of lipid metabolism-related enzymes Srebf1,Acly,Acc,and Fasn in tumor tissues were significantly downregulated in both the low and high-dose matrine groups.Conclusion:Matrine modulates the lipid metabolism pathway,affects tumor cell lipid metabolism,and exerts antitumor effects.

鸡基质蛋白3基因的生物信息学分析

根据GenBank已发表的鸡基质蛋白3(matrin 3)基因序列,设计合成一对特异性引物,以鸡胚成纤维细胞提取的总RNA反转录产物为模板,通过PCR扩增鸡matrin 3基因并进行克隆与生物信息学分析。测序结果表明:鸡matrin 3基因编码区全长为2 709 bp,共编码902个氨基酸,理论pI为5.79,分子质量约为100.71 ku,分子式为C_(4362)H_(6903)N_(1267)O_(1418)S_(29)。序列分析结果显示:鸡matrin 3蛋白有丰富的二级结构,以无规则卷曲和α-螺旋为主;该蛋白中包含两个锌指结构域和两个RNA识别基序,其中人、小鼠和野猪的4个功能结构域氨基酸位点相同,而在鸡matrin 3蛋白中存在多个氨基酸位点变异。核苷酸同源性及遗传进化分析发现:鸡与火鸡的matrin 3基因核苷酸同源性最高(为98.0%),并且与火鸡的亲缘关系最近。研究结果为进一步研究鸡matrin 3蛋白的生物学功能奠定了基础。

Antitumor effect of matrine in human hepatoma G2 cells by inducing apoptosis and autophagy

AIM: To study the antitumor effect of matrine in human hepatoma G2 (HepG2) cells and its molecular mechanism involved in antineoplastic activities. METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect viability of HepG2 cells. The effect of matrine on cell cycle was detected by flow cytometry. Annexin-V-FITC/PI double staining assay was used to detect cellular apoptosis. Cellular morphological changes were observed under an inverted phase contrast microscope. Transmission electron microscopy was performed to further examine ultrastructural structure of the cells treatedwith matrine. Monodansylcadaverine (MDC) staining was used to detect autophagy. Whether autophagy is blocked by 3-methyladenine (3-MA), an autophagy inhibitor, was evaluated. Expression levels of Bax and Beclin 1 in HepG2 cells were measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR).RESULTS: Matrine signif icantly inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner, and induced G1-phase cell cycle arrest and apoptosis of HepG2 cells in a dose-dependent manner. The total apoptosis rate was 0.14% for HepG2 cells not treated with matrine. In contrast, the apoptosis rate was 28.91%, 34.36% and 38.80%, respectively, for HepG2 cells treated with matrine at the concentration of 0.5, 1.0 and 2.0 mg/mL. The remarkable morphological changes were observed under an inverted phase contrast microscope. Abundant cytoplasmic vacuoles with varying sizes were observed in HepG2 cells treated with matrine. Furthermore, vacuolization in cytoplasm progressively became larger and denser when the concentration of matrine was increased. Electron microscopy demonstrated formation of abundant autophagic vacuoles in HepG2 cells after matrine treatment. When the specif ic autophagic inhibitor, 3-MA, was applied, the number of autophagic vacuoles greatly decreased. MDC staining showed that the fluorescent density was higher and the number of MDC-labeled particles in HepG2 cells was greater in matrine treatment group than in control group. Fewer autophagic vacuoles were observed in the combined 3-MA and matrine treatment group when 3-MA was added before matrine treatment, indicating that both autophagy and apoptosis are activated when matrine-induced death of hepatoma G2 cells occurs. Real-time quantitative RT-PCR revealed that the expression levels of Bax gene, an apoptosis-related molecule, and Beclin 1 gene which plays a key role in autophagy were higher in matrine treatment group than in control group, indicating that Beclin 1 is involved in matrineinduced autophagy and the pro-apoptotic mechanismof matrine may be related to its upregulation of Bax expression. CONCLUSION: Matrine has potent antitumor activities in HepG2 cells and may be used as a novel effective reagent in treatment of hepatocellular carcinoma.

Matrine inhibits bladder cancer cell growth and invasion in vitro through PI3K/AKT signaling pathway:An experimental study

Objective:To study the inhibitory effect of matrine on bladder cancer cell growth and invasion in vitro through PI3K/AKT signaling pathway. Methods:Human T24 bladder cancer cell lines were cultured and treated with different doses of matrine(0.25 mg/mL,0.5 mg/mL and 1.0 mg/mL) as well as 20 μmol/L PI3K inhibitor LY294002 for 24 h,and the cell proliferation activity,the number of invasive cells as well as the expression of p-PI3K,p-AKT,proliferation genes and invasion genes were determined. Results:Different doses of matrine could decrease the cell viability value,the number of invasive cells as well as the expression of p-PI3K,p-AKT,MMP2 and MMP9,and increase the expression of p16,p21 and p27 in dose-dependent manner; p16,p21 and p27 expression in cells of 20 μmol/L LY29002 group were significantly higher than those of 0 μmol/L LY29002 group while MMP2 and MMP9 expression were significantly lower than those of 0 μmol/L LY29002 group(P<0.05). Conclusions:Matrine can inhibit bladder cancer cell proliferation and invasion in vitro and regulate the expression of cell cycle-inhibiting molecules and invasion-related genes through PI3K/AKT signaling pathway.

Synergistic protection of astragalus polysaccharides and matrine against ulcerative colitis and associated lung injury in rats

BACKGROUND Ulcerative colitis(UC)is a main form of inflammatory bowel disease.Due to complicated etiology and a high rate of recurrence,it is quite essential to elucidate the underlying mechanism of and search for effective therapeutic methods for UC.AIM To investigate the effects of astragalus polysaccharides(APS)combined with matrine on UC and associated lung injury.METHODS UC was induced in rats by colon mucosal tissue sensitization combined with trinitro-benzene-sulfonic acid-ethanol.Then,the effects of the treatments of salazopyrine,APS,matrine,and APS combined with matrine on histopathological changes of lung and colon tissues,disease activity index(DAI),colon mucosal damage index(CMDI),serum endotoxin(ET)level,serum diamine oxidase(DAO)activity,the contents of tumor necrosis factor-αand interleukin-1β,and the activities of myeloperoxidase,superoxide dismutase,and malondialdehyde in lung tissues,as well as the protein expression of zonula occludens(ZO)-1,Occludin,and trefoil factor 3(TFF3)were detected in UC rats.RESULTS The treatments of salazopyrine,APS,matrine,and APS combined with matrine reduced DAI scores and improved histopathological changes of colon and lung tissues,as well as decreased CMDI scores,ET levels,and DAO activities in UC rats.Moreover,in lung tissues,inflammatory response and oxidative stress injury were relieved after the treatments of salazopyrine,APS,matrine,and APS combined with matrine in UC rats.Furthermore,the expression of ZO-1,Occludin,and TFF3 in lung and colon tissues was increased after different treatments in UC rats.Notably,APS combined with matrine exerted a better protective effect against UC and lung injury compared with other treatments.CONCLUSION APS combined with matrine exert a synergistic protective effect against UC and lung injury,which might be associated with regulating TFF3 expression.

Anti-tumor effect of matrine combined with cisplatin on rat models of cervical cancer

Objective:To observe the anti-tumor effect of matrine combined with cisplatin on U14 rat models of cervical cancer.Methods:A total of 80 female Kunming rats were used to establish U14 rat models of cervical cancer and then divided into groups Ⅰ,Ⅱ,Ⅲ and Ⅳ,with 20 rats in each.For Group Ⅰ,the control group,injection of normal saline was given around the tumors.For Group Ⅱ,injection of 2 mg/kg cisplatin was given around the tumors.For Group Ⅲ,injection of 75 mg/kg matrine was given around the tumors while the combined injection of matrine and cisplatin was given for Group Ⅳ with the same doses as Groups Ⅱand Ⅲ.The animals were sacrificed 10 d after the injection and tumors were taken out for the comparisons of tumor weights after injection and calculation of anti-tumor rates,while thymus and spleen were taken for thymus index and spleen index.Blood in eyeball was collected for determination of changes in serum creatinine and urea nitrogen levels.Sections of tumor issue were prepared and morphological changes in tumor tissue cells were observed by using immunohistochemistry technique.Results:After injection,the thymus index and spleen index in Groups Ⅲ and Ⅳ were significantly higher than those in Groups Ⅰ and Ⅱ(P<0.05)while the two indexes in Group Ⅱ were significantly lower than Group Ⅰ(P<0.05).The tumor weights in Groups Ⅱ and Ⅳ were significantly smaller than those in Groups Ⅰ and Ⅲ(P<0.05) with significantly higher anti-tumor rates than Groups Ⅰ and Ⅲ(P<0.05).The serum creatinine and urea nitrogen levels in Groups Ⅲ and Ⅳ were significantly lower than Group Ⅱ(P<0.05) and the two indicators in Group Ⅲ were significantly lower than those in Group Ⅳ(P<0.05).The observation under the histological microscope showed densely arranged tumor cells in Group Ⅰ,growing as a crumby structure and diffuse appearance,with hyperchromatic and large nuclei,and abundant cytoplasm.In the case of Group Ⅱ,it showed less tumor cells,with extensive degenerative necrosis,sparse arrangement and karyopyknosis as well as karyoclasis.For Group Ⅲ,necrosis of tumor cells in different sizes and heterogeneous color in nuclei were observed.For Group Ⅳ,the number of tumor cells was significantly smaller than Groups Ⅰ and Ⅲ and the tumor cells presented an appearance of crumby structure as cancer nests,with more proliferation of connective tissue.Conclusions:The treatment of matrine combined with cisplatin can significantly improve the anti-tumor effect on U14 rats with cervical cancer,which can be a new option for the treatment for cervical cancer.

Effect of matrine on transforming growth factor β1 and hepatocyte growth factor in rat liver fibrosis model

Objective:To observe the preventive and control effect of matrine on transforming growth factor(TCF- β1) and hepatocyte.growth factor(HCF) of liver fibrosis tissue in rals.Methods:A total of48 SD rats were randomly divided into A,B,C,D groups with 12 in each,group A as the normal control group and groups B.C,D as liver fibrosis models using composite modulus method with carbon tetrachloride(CCL_4).Group B was the model group,group C adopted γ— interferon lavage therapy in the second day of modeling,and group D adopted matrine lavage treatment,at 4 and8 weeks after treatment.Six rats were executed for detection of TGF- β1 and HGF,liver tissue histology and comparison fibrosis degree changes of rat liver tissue between groups.Results:Croups B,C,D showed a more significantly increased TCF- β1 at each time point compared with group A(P<0.05);Group B showed a more significantly increased TGF- β1 than groups C and D at weeks 4 and 8(P<0.05);group D showed a lowest level of TGF-β1,followed by groups C and B.HGF of group B decreased more significantly than A group at weeks 4 and 8(P<0.05);HGF of groups C and D was significantly elevated at 4 and 8 weeks than groups A and B(P<0.05),in which the group D showed the highest level of HGF.According to tissue histologic observation,rat liver tissue structure of group A was clear and normal,tissue structure of group B was destroyed with obvious fibrous tissue hyperplasia and fatty change of hepatic cells;groups C and D showed a slighter liver tissue damage,cell necrosis and connective tissue hyperplasia in collect abbacy than group B with a trend of obvious improvement.Conclusions:Matrine can reduce TGF- β1expression and enhance the activity of HGF,so as to realize the inhibition effect on liver fibrosis in rats.

Efficacy of intramuscular matrine in the treatment of chronic hepatitis B

BACKGROUND: Hepatitis B virus (HBV) infection, a glo- bal public health problem, is the leading cause of cirrhosis and hepatocellular carcinoma (HCC) worldwide. There are more than 350 million HBV carriers in the world and up to one million die annually due to hepatitis B associated liv- er disease. So far no optimal treatment is available for pa- tients with chronic hepatitis B. In the paper we investigated the efficacy of intramuscular matrine in the treatment of chronic hepatitis B. METHODS: One hundred and twenty patients with chronic hepatitis B were randomly divided into matrine treatment group (n =60) and control group (n =60). The patients of the matrine group were given intramuscularly with matrine (an alkaloid extracted from a traditional Chinese herb Radix Sophorae Flavescentis by Guangzhou Ming Xing Pharmaceu cal Factory, Guangzhou, China) of 100 mg daily for 90 days in addition to conventional liver-protective drugs in- cluding glucurone, inosine, compound vitamin B and caryophyllin. The control group received conventional liv- er-protective drugs alone. Clinical manifestations and labo- ratory parameters including liver biochemistry and serum hepatitis B virus markers were monitored before and after treatment in the two groups. RESULTS: Significant differences were seen between the two groups in terms of improvement of clinical symptoms and signs, recovery of liver functions, and serum conver- sion from hepatitis Be antigen to HBe antibody and from positive to negative serum HBV DNA (P <0.05-0.01). The result of the matrine group was more marked than that of the control group. Serious side-effects were not observed except mild pain at the site of injection of matrine in a few patients. CONCLUSION: These results indicate that intramuscular matrine may be an economical, efficacious, safe drug for the treatment of chronic hepatitis B.

Relaxant Effects of Matrine on Aortic Smooth Muscles of Guinea Pigs

Objective To determine whether matrine, a kind of traditional Chinese medicinal alkaloid, can relax the aortic smooth muscles isolated from guinea pigs and to investigate the mechanism of its relaxant effects. Methods Phenylephrine or potassium chloride concentration-dependent relaxation response of aortic smooth muscles to matrine was studied in the precontracted guinea pigs. Results Matrine （1×10^-4 mol/L -3.3×10^-3 mol/L） relaxed the endothelium-denuded aortic rings pre-contracted sub-maximally with phenylephrine, in a concentration-dependent manner, and its pre-incubation （3.3× 10^- 3 mol/L） produced a significant rightward shift in the phenylephrine dose-response curve, but had no effects on the potassium chloride-induced contraction. The anti-contractile effect of matrine was not reduced by the highly selective ATP-dependent K^＋ channel blocker glibenclamide （10.5 mol/L）, either by the non-selective K^＋channel blocker tetraethylammonium （10^-3 mol/L）, or by the β-antagonist propranolol （10^-5 mol/L）. In either ＂normal＂ or ＂Ca^2＋-free＂ bathing medium, the phenylephrine-induced contraction was attenuated by matrine （3.3×10^-3 mol/L）, indicating that the vasorelaxation was due to inhibition of intracellular and extracellular Ca^2＋ mobilization. Conclusion Matrine inhibits phenylephrine-induced contractions by inhibiting activation of α-adrenoceptor and interfering with the release of intracellular Ca^2＋ and the influx of extracellular Ca^2＋.

Matrine promotes neural circuit remodeling to regulate motor function in a mouse model of chronic spinal cord injury

In chronic phase of spinal cord injury, functional recovery is more untreatable compared with early intervention in acute phase of spinal cord injury. In the last decade, several combination therapies successfully improved motor dysfunction in chronic spinal cord injury. However, their effectiveness is not sufficient. We previously found a new effective compound for spinal cord injury, matrine, which induced axonal growth and functional recovery in acute spinal cord injury mice via direct activation of extracellular heat shock protein 90. Although our previous study clarified that matrine was an activator of extracellular heat shock protein 90, the potential of matrine for spinal cord injury in chronic phase has not been sufficiently evaluated. Thus, this study aimed to investigate whether matrine ameliorates chronic spinal cord injury in mice. Once daily intragastric administration of matrine(100 μmol/kg per day) to spinal cord injury mice were starte at 28 days after injury, and continued for 154 days. Continuous mat rine treatment improved hindlimb motor function in chronic spinal cord injury mice. In injured spinal cords of the matrine-treated mice, the density of neurofilament-H-positive axons was increased. Moreover, matrine treatment increased the density of bassoon-positive presynapses in contact with choline acetyltransferase-positive motor neurons in the lumbar spinal cord. These findings suggest that matrine promotes remodeling and reconnection of neural circuits to regulate hindlimb movement. All protocols were approved by the Committee for Animal Care and Use of the Sugitani Campus of the University of Toyama(approval No. A2013 INM-1 and A2016 INM-3) on May 7, 2013 and May 17, 2016, respectively.

Inhibitory Effect of Matrine on the Expression of PSA and AR in Prostate Cancer Cell line LNCaP

In order to investigate the inhibitory effect of matrine on the expression of prostate specific antigen （PSA） and androgen receptor （AR） in prostate cancer cell line LNCaP in vitro, LNCaP cells were treated with matrine at different concentrations （0.5, 1.0, 1.5, 2.0 g/L） for 12-36 h. The growth activities of cancer cells were determined by MTT colorimetric assay. The AR level was measured by Western blotting. The expression of PSA was detected by using AXSYM system-chemical luciferase methods. The results showed that matrine could effectively inhibit the growth of androgen-dependent prostate cancer cell line LNCaP in vitro in a time-and dose-dependent manner （P〈0.05）. It could obviously decrease the level of AR （P〈0.01） and inhibit the expression of PSA in a dose-dependent manner （P〈0.05） in LNCaP cells. It was concluded that matrine could significantly suppress the growth of LNCaP cells and inhibit the expression of PSA and AR of prostate cancer cells.

Matrine inhibits mycelia growth of Botryosphaeria dothidea by affecting membrane permeability

Matrine is a promising botanical antifungal;however, the mechanism underlying the antifungal activity is yet limited. We studied the antifungal activity of matrine and the underlying mechanism in Botryosphaeria dothidea as a model strain. Matrine strongly inhibited mycelial growth of B. dothidea in a dose-dependent manner. Matrine-treated B. dothidea showed morphological and ultrastructural alterations, including shriveled hyphae, plasmolysis, and leakage of cytoplasm related to cell membrane deterioration. In addition, matrine caused significantly high conductivity and absorbance (260 nm) in extracellular matrices and low lipid contents in B. dothidea, indicating increased membrane permeability. Lipid peroxidation showed that matrine resulted in increased malondialdehyde content while enhancing the generation of reactive oxygen species and the activities of superoxide dismutase, catalase, and peroxidase. These results showed that matrine inhibited the mycelial growth of B. dothidea by enhancing cell membrane permeability via membrane lipid peroxidation.

Antifungal activities of matrine and oxymatrine and their synergetic effects with chlorthalonil

The EC50 values of matrine and oxymatrine against five forest pathogenic fungi （Fusarium oxysporum, Valsa pini, Cladosporium oxysporum, Sphaeropsis sapinea, Marssonina brunnea） were examined by bioassay methods. The results demonstrated that matrine and oxymatrine had strong inhibitory activities to the conidium germination of the tested fungi. The ECso values of matrine for inhibiting the conidium germination of Marssonina brunnea, Cladosporium oxysporum, Sphaeropsis sapinea were 123μg·mL^-1, 272 μg·mL^-1, 1133 μg·mL^-1, respectively, and the EC50 values of oxymatrine for inhibiting the conidium germination of Fusarium oxysporum, Sphaeropsis sapinea were 532μg·mL^-1, 601μg·mL^-1, respectively. The hyphal growth of the fungi was also significantly inhibited by matrine and oxymatrine. The ECs0 values of matrine inhibiting the conidium germination of Sphaeropsis sapinea, Valsa pini, Fusarium oxysporum were 428μg·mL^-1, 535 μg·mL^-1, 592 μg·mL^-1, respectively. The EC50 values of oxymatrine inhibiting the conidium germination of Valsa pini, Fusarium oxysporum were 323, 618μg·mL^-1, respectively. In the synergetic tests the ECs0 values of the mixtures of thiophanate methyl （or chlorthalonil） and matrine （or oxymatrine） were lower than 34 μg·mL^-1 while their co-toxicity coefficients were significantly higher than 1130 It indicated that the mixture of the alkaloids and the chemical had potential practical utilization in controlling certain forest fungal diseases.

Compound Kushen injection induces immediate hypersensitivity reaction through promoting the production of platelet-activating factor via de novo pathway

OBJECTIVE Compound Kushen injection(CKI)is a bis-herbal formulation extracted from Kushen(Radix Sophorae Flavescentis)and Baituling(Rhizoma Heterosmilacis Japonicae).Clinically,it is used as the adjuvant treatment of cancer.However,with the increased application,the cases of immediate hypersensitivity reactions(IHRs)also gradually rise.In this study,we investigated the underlying mechanism(s)and active constituent(s)for CKI-induced IHRs in experimental models.METHODS T helper 2(Th2)immunity-amplified mice were prepared by aluminum adjuvant.Anaphylactic shock was detected by measuring rectal thermometry in propranolol pretreated mice.For evaluating microvascular permeability,Evans blue extravasation assay was used.Platelet-activating factor(PAF),serum total IgE(tIgE)and mouse mast cell protease 1(MMCP1)were measured by ELISA.RESULTS The obtained results showed that CKI did not elevate serum tIgE and MMCP1 after consecutive immunization for five weeks,but could induce Evans blue extravasation(local)and cause obvious hypothermia(systemic)after a single injection.Further study showed that alkaloids in Kushen,especially matrine,were responsible for CKI-induced IHRs.Mechanism study showed that various PAF receptor antagonists could significantly counter CKI-induced IHRs locally or systemically.In cell system,CKI was able to promote PAF production in a non-cell-selective manner.In cell lysate,the effect of CKI on PAF production became stronger and could be abolished by blocking de novo pathway.CONCLUSION In conclusion,our study identifies,for the first time,that CKI is a PAF inducer.It causes non-immunologic IHRs,rather than IgE-dependent IHRs,by promoting PAF production through de novo pathway.Alkaloids in Kushen,especially matrine,are the prime culprits for IHRs.Our findings may provide a potential approach for preventing and treating CKI-induced IHRs.

Inhibitory effects of matrine on electrical signals and amino acid neurotransmitters in hippocampal brain slices

BACKGROUND： Studies on electrical signals of hippocampal brain slices in vivo have shown that matrine inhibits benzylpenicillin sodium-induced activation of neuronal signal transduction. OBJECTIVE： To verify the inhibition effect of matrine on activation of electrical signals in rat brain slices and the role rnatrine plays in hippocampal amino acid transmitter release. DESIGN, TIME AND SETTING： The in vitro, neurophysiological, controlled experiment was performed in the Zhejiang Province Key Laboratory of Cardio-cerebrovascular Disease and Nerve System Drugs Appraisement and Chinese Traditional Medicine Screening and Research between July 2003 and May 2004. The in vivo, neuronal, biochemical experiment was performed in the Zhejiang Province Key Laboratory of Chinese Traditional Medicine Quality Standardization from July 2005 to December 2006. MATERIALS： Forty healthy, Sprague Dawley rats, 7-8 weeks old, and 120 healthy, ICR mice, 5-6 weeks old, were included in this study, irrespective of gender. Matrine powder was provided by the National Institute for the Control of Pharmaceutical and Biological Products, China. Matrine injection was purchased from Zhuhai Biochemical Pharmaceutical Factory, China. Penicillin was bought from Shijiazhuang Pharmaceutical Group Co., Ltd., China. METHODS： （1） Rats were randomly assigned to four groups： control, penicillin model, and matrine high-dose and low-dose, with 10 rats in each group. The control group was perfused with artificial cerebrospinal fluid. In the remaining three groups, hippocampal brain slices were perfused with normal artificial cerebrospinal fluid containing 1 × 106 U/L penicillin for the first 10 minutes. The penicillin model group received artificial cerebrospinal fluid for an additional 30 minutes, while the matrine high-dose and low-dose groups received 0.1 g/L and 0.05 g/L matrine, respectively, for an additional 30 minutes. （2） Mice were randomly assigned to four groups （n = 30）. The matrine high-, medium-, and low-dose groups were separately injected with 58.5, 39.0, and 19.5 mg/kg matrine via caudal vein, respectively. No intervention was administered to the normal group. MAIN OUTCOME MEASURES： The field potential value in the CA1 region of penicillin-induced rat hippocampal brain slices was analyzed using the evoked field potential technique; chromatography was utilized to determine y-aminobutyric acid and glutamic acid content in the mouse hippocampus. RESULTS： （1） Both 0.1 g/L and 0.05 g/L matrine reduced the number of evoked field potentials in the penicillin-induced rat hippocampal brain slices （P 〈 0.05 or P 〈 0.01 ）. In addition, 0.1 g/L matrine led to a reduction of evoked field potential amplitude （P 〈 0.05）. （2） Compared with normal mice, γ-aminobutyric levels were dramatically increased at 20 minutes after high-dose matrine treatment （P 〈 0.05）. In addition, significantly increased γ-aminobutyric acid levels were observed at 40 minutes after medium- and low-dose matrine treatments （P 〈 0.05 or P 〈 0.01 ）. The glutamic acid/γ-aminobutyric acid ratio was significantly less at 20 minutes after high-dose matrine treatment compared with the normal mice group （P 〈 0.05）. CONCLUSION： Matrine exerts a central inhibitory effect via increased inhibitory neurotransmitter γ-aminobutyric acid levels in the hippocampus.