Effect of matrine on apoptosis of nucleus pulposus cells in rats with intervertebral disc degeneration / 中国组织工程研究

BACKGROUND:Nucleus pulposus cell apoptosis is the main pathological basis for intervertebral disc degeneration,and inflammation and peroxidation are important factors leading to apoptosis in the nucleus pulposus.Studies have shown that matrine has antioxidant,senescent,inflammatory and apoptotic effects,and may be a potential drug for the treatment of disc degeneration. OBJECTIVE:To investigate the effect of matrine on apoptosis of nucleus pulposus cells in rats with intervertebral disc degeneration by regulating the cyclic GMP-AMP synthase(cGAS)-stimulator of interferon genes(STING)signaling pathway. METHODS:(1)Nucleus pulposus cells of rats at a logarithmic phase were randomly separated into a control group,a model group,a low-dose matrine group,a high-dose matrine group,an empty group,and a high-dose matrine+cGAS overexpression group.Except for the control group,cell models of intervertebral disc degeneration were established in the other groups through oxygen-glucose deprivation.At the same time of modeling,the low-dose and high-dose groups were treated with 0.4 and 0.8 mmol/L matrine,respectively,and the empty group was transfected with the empty plasmid,while the high-dose+cGAS overexpression group was treated with 0.8 mmol/L matrine with the transfection of the cGAS overexpression plasmid.After 24 hours of treatment,cell activity and apoptosis,intracellular levels of reactive oxygen species,superoxide dismutase,tumor necrosis factor α and interleukin 1β,and intracellular expression of apoptotic proteins and cGAS-STING pathway proteins were detected.(2)Sixty Sprague-Dawley rats were randomized into six groups(n=10 per group):control group,model group,low-dose matrine group,high-dose matrine group,empty group,and high-dose+cGAS overexpression group.After 12 weeks of modeling,60 and 120 mg/kg matrine were given by gavage in the low-dose and high-dose matrine groups,respectively(once a day),and the empty plasmid was injected into the tail vein in the empty group(2 times/week),while the high-dose+cGAS overexpression group was given 120 mg/kg matrine by gavage and injected with cGAS overexpression plasmid to the tail vein.Treatment in each group was given consecutively for 3 weeks.Samples were taken after drug administration and assayed for apoptosis,levels of reactive oxygen species,superoxide dismutase,tumor necrosis factor α and interleukin 1β,as well as apoptotic protein and cGAS-STING pathway protein expression. RESULTS AND CONCLUSION:Compared with the control group,in the model group,cell activity and superoxide dismutase levels were decreased(P<0.05),and apoptosis rate,levels of reactive oxygen species,tumor necrosis factor α and interleukin 1β,and the expression of cGAS,STING,cleaved caspase-3 and Bax proteins were elevated(P<0.05).Matrine dose-dependently ameliorated the above changes in each index due to cellular modeling(P<0.05),whereas cGAS overexpression partially antagonized the ameliorative effect of high-dose matrine.Similar results to the in vitro cellular experiments were obtained in animal experiments.These results indicate that matrine could inhibit inflammation and oxidative stress by blocking the cGAS-STING signaling,which in turn attenuates apoptosis and elevates the activity of nucleus pulposus cells in rats with intervertebral disc degeneration.

Impact of Matrine on Th17/Treg Cell Balance in Coronary Heart Disease Rats by Regulating the RhoA-ROCK Signaling Pathway / 中药新药与临床药理

Objective To investigate the impacts of matrine on the balance of helper T cell 17(Th17)/regulatory T cell(Treg)and the Ras homolog gene family member A(RhoA)-Rho-associated coiled-coil forming protein kinase(ROCK)signaling pathway in coronary heart disease(CHD)rats.Methods A model of coronary heart disease was established.Rats were grouped into control group,model group(CHD group),low-dose matrine(50 mg·kg-1,Matrine-L)group,high-dose matrine(200 mg·kg-1,Matrine-H)group,and Matrine-H+LPA(200 mg·kg-1 matrine+10 mg·kg-1 LPA)group.Echocardiography was applied to detect cardiac function.Enzyme linked immunosorbent assay(ELISA)method was used to detect interleukin-17(IL-17)and transforming growth factor(TGF-β).The quantity of Th17,Treg and Th17/Treg ratio were detected by flow cytometry.Immunohistochemistry was applied to detect the protein expressions of endothelial nitric oxide synthase(eNOS)and endothelin 1(ET-1).Masson staining was carried out to observe the pathological changes of myocardial tissue.The myocardial infarction in each group of rats was observed by TCC staining.TUNEL staining was performed to detect cell apoptosis in myocardial tissue.Additionally,RhoA activity was detected by assay kit.Western Blot method was applied to detect the protein expressions levels of B-cell lymphoma factor 2(Bcl-2),Bcl-2 associated X protein(Bax),cysteine aspartate proteinase-3(Caspase-3),RhoA,ROCK1 and ROCK2.Results Compared with the control group,a large amount of blue collagen fiber deposition was observed in the myocardial tissue of CHD group.The expression levels of left ventricular end-diastolic volume(LVEDV),left ventricular end-systolic volume(LVESV),IL-17,Th17,Th17/Treg,ET-1,infarct size,cell apoptosis rate,TUNEL positive rate,Bax,Caspase-3,RhoA activity,RhoA,ROCK1,ROCK2 were obviously increased.The expression levels of left ventricular ejection fraction(LVEF),left ventricular shortening fraction(LVFS),TGF-β,Treg,eNOS,and Bcl-2 were obviously reduced(P＜0.05).Compared with the CHD group,blue collagen fibers in myocardial tissue of Matrine-L and Matrine-H groups gradually decreased.The expression levels of LVEDV,LVESV,IL-17,Th17,Th17/Treg,ET-1,infarct size,cell apoptosis rate,TUNEL positive rate,Bax,Caspase-3,RhoA activity,RhoA,ROCK1 and ROCK2 were obviously reduced in sequence.The expression levels of LVEF,LVFS,TGF-β,Treg,eNOS,and Bcl-2 were also obviously increased in sequence(P＜0.05).Compared with the Matrine-H group,blue collagen fibers in myocardial tissue of Matrine-H+LPA group increased.The expression levels of LVEDV,LVESV,IL-17,Th17,Th17/Treg,ET-1,infarct size,cell apoptosis rate,TUNEL positive rate,Bax,Caspase-3,RhoA activity,RhoA,ROCK1,ROCK2 were obviously increased,while the expression levels of LVEF,LVFS,TGF-β,Treg,eNOS and Bcl-2 were obviously reduced(P＜0.05).Conclusion Matrine regulates Th17/Treg cell balance and improves myocardial injury in rats with CHD by inhibiting the RhoA-ROCK signaling pathway.

Improvement effect and mechanism of matrine on experimental autoimmune encephalomyelitis mice / 中国药房

OBJECTIVE To investigate whether matrine exerts improvement effect on experimental autoimmune encephalomyelitis （EAE） mice by regulating ferroptosis pathway. METHODS Totally 30 female C57BL/6 mice were randomly assigned into normal group， model group and matrine group， with 10 mice in each group. Model group and matrine group were given antigen emulsion containing inactivated Mycobacterium tuberculosis and MOG35-55 to induce EAE model. Matrine group was injected with Matrine injection （50 mg/kg） intraperitoneally since the 7th day after immunization； normal group and model group were given constant volume of normal saline intraperitoneally， once a day， since 18th day after immunization. The neurofunctional score of mice was recorded， and hematoxylin and eosin staining and Luxol fast blue staining were used to observe inflammatory cell infiltration and demyelination in spinal cord tissue. The quantitative reverse transcription PCR and Western blot assay were performed to determine the mRNA expressions of transferrin receptor 1 （TFR1）， nuclear receptor coactivator 4 （NCOA4） and hephaestin （Heph）， and the protein expressions of system Xc－ （xCT） and glutathione peroxidase 4 （GPx4）. RESULTS Compared with normal group， accumulative neurofunctional score was significantly increased in model group （P＜0.01）； inflammatory cell infiltration and demyelination were obvious in spinal cord tissue， and related scores were increased significantly （P＜0.01）. The mRNA expressions of TFR1 and NCOA4 in myelin tissue were up-regulated significantly， while the mRNA expression of Heph and the protein expressions of xCT and GPx4 were down-regulated significantly （P＜0.05 or P＜0.01）. Compared with model group， above indexes of matrine group were all improved significantly （P＜0.05 or P＜0.01）. CONCLUSIONS Matrine can improve EAE mice， the mechanism of which may be associated with regulating iron metabolism pathway and xCT/GPx4 pathway in ferroptosis.

Effect and mechanism of matrine on steatosis Chang Liver cells induced by oleic acid / 中国药房

OBJECTIVE To investigate the effects of matrine （MT） on steatosis Chang Liver cell model induced by oleic acid （OA） and its possible mechanism. METHODS Chang Liver cells were divided into blank group， model group and MT low-dose， medium-dose group and high-dose groups （0.1， 0.5， 1.0 mmol/L）. Except for blank group， the other groups were treated with 1.0 mmol/L OA for 24 h to establish steatosis model， and MT groups were given corresponding concentrations of drugs for 24 h. The activities of steatosis Chang Liver cells were observed； the morphologies of intracellular lipid droplets were observed and lipid content was also determined. The contents of liver function indexes [alanine transaminase （ALT）， aspartate transaminase （AST）， total bilirubin （TBIL）， alkaline phosphatase （ALP）]， as well as mRNA and protein expressions of farnesoid X receptor （FXR）， cytochrome P450 7A1 （CYP7A1） and fibroblast growth factor 19 （FGF19） were all detected. RESULTS OA and MT had no significant effect on the activity of Chang Liver cells. After OA treatment， orange lipid droplets formed in cytoplasm； compared with blank group， relative lipid content and the levels of liver function indexes were increased significantly， while the mRNA and protein expressions of FXR， CYP7A1 and FGF19 were down-regulated significantly （P＜0.05）. After treatment of low， medium and high concentrations of MT， above indexes were all reversed significantly （P＜0.05）. CONCLUSIONS MT could significantly improve the lipid content and liver function indexes of steatosis Chang Liver cells induced by OA though regulating FXR/CYP7A1/ FGF19 signaling pathway.

Influence of matrine on Th17/Treg balance in rats with inflammatory bowel disease by regulating IL-6/STAT3/NF-κB signaling pathway / 西安交通大学学报(医学版)

【Objective】 To investigate the influence of matrine (MT) on the balance of T helper cell 17 (Th17)/regulatory T cells (Treg) in rats with inflammatory bowel disease by regulating interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3)/nuclear transcription factor-κB (NF-κB) pathway. 【Methods】 SD rats were grouped into control check group (CK group), model group, low-dose MT group (MT-L group, 50 mg/kg), medium-dose MT group (MT-M group, 100 mg/kg), high-dose MT group (MT-H group, 200 mg/kg), mesalazine group (MSLM group, 0.42 g/kg), and MT-H+rIL-6 (IL-6 activator) group (200 mg/kg+0.05 mg/kg) according to the random number table method, with 18 in each group. Except for the CK group, the rats in other groups all received with 5% trinitrobenzenesulfonic acid (20 mg/kg) buffer solution mixed with 50% ethanol at a ratio of 1∶1 and then enema to construct a rat model of inflammatory bowel disease. After the successful modeling, they were treated with drug administration once a day for 7 weeks. The body weight of rats was measured at 1, 3, 5, and 7 weeks of administration; the changes of colon length of rats in each group were compared; HE staining was used to detect the pathological damage of rat colon tissue; the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-17 and IL-10 in serum of rats were detected by ELISA; the proportions of Th17 and Treg cells in peripheral blood of rats were detected by flow cytometry; Western blottingt was performed to detect the protein expression of retinoic acid-related orphan receptor γt (RORγt), forkhead box protein P3 (Foxp3), IL-6, p-STAT3, and p-NF-κB p65 in rat colon tissue. 【Results】 Compared with the CK group, the colon tissue of the model group was severely damaged by pathology, and the body weight (at 3, 5, and 7 weeks), the level of IL-10, the proportion of Treg cell, and the expression of Foxp3 protein were decreased, the colon length shortened, the levels of TNF-α, IL-17, the proportions of Th17 cell, Th17/Treg ratio, and the protein expression of RORγt, IL-6, p-STAT3, and p-NF-κB p65 increased (P<0.05). Compared with the model group, the corresponding indicators of the MT-L group, MT-M group, MT-H group, and MSLM group had the opposite trends (P<0.05); rIL-6 attenuated the promoting effect of high-dose MT on Th17/Treg balance in inflammatory bowel disease rats. 【Conclusion】 MT may promote Th17/Treg balance in inflammatory bowel disease rats by inhibiting IL-6/STAT3/NF-κB signaling pathway.

Molecular mechanism of Matrine Injection in treating colorectal cancer based on network pharmacology and molecular docking / 国际生物医学工程杂志

Objective:To explore the molecular mechanism of Matrine Injection in treating colorectal cancer based on network pharmacological analysis and molecular docking.Methods:Taking matrine as the object, the corresponding potential drug targets in matrine were obtained from Swiss Target Prediction database, and SuperPred database, and the database of traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP). Differential genes were obtained from gene expression omnibus (GEO), and GeneCards, OMIM, DrugBank, and CTD databases were used to collect colorectal cancer-related genes. Furthermore, core targets were screened by establishing protein-protein interaction (PPI) networks. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis were performed by bioconductor of R language. Finally, the molecular docking calculation was performed to evaluate the interaction between matrine and core targets.Results:Matrine contained 63 targets. A total of 14 198 targets for colorectal cancer were obtained. The topology analysis results of the PPI network showed that 5 main targets such as myelocytomatosis proteins (MYC), interleukin-6 (IL-6), Caspase-3 (CASP3), mammalian target of rapamycin (mTOR), and amphiregulin (AR). GO enrichment analysis found that biological process (BP) mainly includes hydrogen peroxide reaction, cell reaction to hydrogen peroxide and cell response to chemical stress, etc; Cell components (CC) mainly include lipid rafts, membrane microregions and synaptic membranes, etc; Molecular functions (MF) mainly include transcriptional coregulatory factor binding, postsynaptic neurotransmitter receptor activity and core promoter sequence-specific DNA binding. KEGG pathway analysis showed that it involved chemical carcinogenesis-receptor activation, tumor necrosis factor (TNF) signaling pathway, and Toll-like receptor signaling pathway, etc. Molecular docking showed that matrine had good binding with the core target.Conclusions:Matrine acts on targets such as MYC, IL-6, CASP3, mTOR, and AR, and exerts therapeutic effects on colorectal cancer by regulating chemical carcinogenesis-receptor activation, TNF signaling pathway, Toll-like receptor signaling, etc.

Matrine treats allergic asthma through inhibition of Lyn/Syk signaling pathway in rat's model / 免疫学杂志

To investigate the impact of matrine(Mat)on the immune function of rats with allergic asthma by regulating the Lyn/Syk signal pathway,12 SD rats were randomly selected as control group(NC group),and the other rats were modeled by ovalbumin(OVA)with reference to previous literature.The successfully modeled allergic asthma rats were randomly grouped into model group,Mat group(100 mg/kg Mat),MLR-1023 group(30 mg/kg Lyn/Syk signal pathway activator MLR-1023),and Mat+MLR-1023 group(100 mg/kg Mat+30 mg/kg MLR-1023),with 12 rats in each group.NC group and Model group were given the same amount of physiological saline once a day for 4 weeks.Bronchoalveolar lavage fluid(BALF)were collected and eosinophils were counted;the level of cytokines in serum and BALF were detected by ELISA kit;the pathological change of lung tissue was evaluated by HE staining;the histamine level in lung tissue was measured;Western blot was applied to detect the levels of Lyn/Syk pathway related proteins.Data showed that the lung tissue in NC group was stained clearly and the structure was normal.In the model group,a large number of inflammatory cells infiltrated,the eosinophils around the bronchus increased,epithelial cells enlarged and fell off.Compared with NC group,the model group demonstrated obvious increase in the number of eosinophils,the levels of TNF-α,IL-4,IL-5,IL-1β and LTD-4 in serum,the levels of IgE and OVA sIgE in BALF,and the levels of histamine,p-Lyn/Lyn and p-Syk/Syk in lung tissue(P<0.05).Matrine could reverse these pathological changes mentioned above in model rats(P<0.05),while MLR-1023 eliminated the therapeutic effect of Mat on allergic asthma rats.Taken together,Mat may improve the immune function of rats with allergic asthma by down-regulating the Lyn/Syk signal pathway.

Effect of matrine on proliferation, apoptosis and radiotherapy sensitivity of uveal melanoma cells / 中华眼底病杂志

Objective:To investigate the effects of matrine on proliferation, apoptosis and radiotherapy sensitivity of uveal melanoma cells.Methods:An animal experiment study. In vitro experiment: MuM2B cells of human choroidal melanoma were randomly divided into control group and matrine 0.25, 0.50, 1.00, 2.00 g/L groups. The cell morphology was observed by transmission electron microscope. Cell proliferation was detected by thiazole blue colorimetry. The mRNA and relative expression levels of CyclinD D (CyclinD), B lymphoblastoma-2 (Bcl-2) and Bcl2-associated X protein (Bax) were detected by real-time polymerase chain reaction and Western blot. In vivo experiment: BALB/C mice were injected with MuM2B cell suspension subcutaneously on the back of forelimb to prepare transplanted tumor model. After successful modeling, they were randomly divided into blank group and matrine treatment group with different concentrations. Mice in blank group were injected with phosphate buffer subcutaneously. Mice in different matrine treatment groups were injected with 15, 25, 50, 100 mg/kg matrine subcutaneously, respectively, for 7 consecutive days. The tumor was weighed and its volume was measured after the last administration. Single factor analysis of variance was used to compare different groups. The t test was used for pairwise comparison between groups. Results:In the control group, the cell structure was normal, the distribution was uniform, and no or rare nuclear pyknosis was seen. With the increase of matrine dosage, the nuclear pyretosis increased gradually and cell morphology changed obviously. Compared with the control group, the cell survival rate in 0.50, 1.00 and 2.00 g/L groups gradually decreased with matrine concentration increasing and treatment time prolongating, the relative expression levels of CyclinD and Bcl-2 mRNA and protein gradually decreased, and the relative expression levels of Bax mRNA and protein gradually increased. Under the same radiation dose X-ray irradiation, the cell survival rate of 0.50, 1.00 and 2.00 g/L groups gradually decreased, and the differences were statistically significant ( P<0.05). Compared with blank group, the tumor weight and volume of mice in different doses of matrine group were significantly decreased, and the differences were statistically significant ( P<0.05). Conclusion:Matrine can down-regulate the expression of CyclinD and Bcl-2, up-regulate the expression of Bax, promote the apoptosis of MuM2B human melanoma cells, inhibit cell proliferation, and enhance cell radiosensitivity.

Study on Mechanism of Action of Matrine in Treatment of Inflammatory Bowel Disease Based on Zebrafish Model and Network Pharmacology / 中国实验方剂学杂志

ObjectiveTo study the mechanism of matrine in the treatment of inflammatory bowel disease （IBD） based on the zebrafish model and network pharmacology. MethodThe IBD model of zebrafish was established using 2，4，6-trinitro-benzenesulfonicacid （TNBS）， and the intestinal phagocytic function， goblet cell secretion， and neutrophil aggregation were evaluated using neutral red staining， alcian blue staining， and neutrophil number changes. Changes in tumor necrosis factor （TNF）-α and cholecystokinin （CCK） content in zebrafish were determined by using relevant reagent kits. Network pharmacology and molecular docking techniques were used to predict the potential mechanism of matrine in the treatment of IBD. Gene expression of relevant targets was verified through Real-time polymerase chain reaction （Real-time PCR）. ResultCompared with the model group， the matrine administration group can increase the neutral red staining area in a dose-dependent manner and improve intestinal phagocytic function（P<0.05，P<0.01）. It can reduce the staining area of alcian blue and affect the secretion of intestinal goblet cells（P<0.01）. It can reduce the number of neutrophil granulocytes， relieve its aggregation， significantly reduce TNF-α content（P<0.01）， and increase the CCK content. Network pharmacology analysis identifies 28 potential targets for matrine in the treatment of IBD. The top five targets by protein-protein interaction （PPI） network analysis are CHRNA7， DRD1， CHRNA4， SLC6A3， and GRM5. The Kyoto encyclopedia of genes and genomes （KEGG） results show that the treatment of IBD with matrine may be related to neuroactive ligand-receptor interaction， cholinergic synapse， and neutrophil extracellular trap formation. Real-time PCR results show that matrine can affect the expression level of related target genes. Conclusionmatrine has a certain therapeutic effect on IBD and can affect the inflammatory response of IBD. Its therapeutic effect may be related to neuroactive ligand-receptor interaction and other pathways.

Synthesis of matrine derivative ZS10 and its mechanism of apoptosis induced by PI3K/AKT signaling pathway in human hepatocellular carcinoma cell line BEL-7402 / 中国药理学通报

Aim To explore the signaling pathway of matrine derivative ZS10 in inhibiting proliferation and inducing apoptosis of BEL-7402 cells. Methods ZS10 was synthesized by organic synthesis. The inhibitory effect of ZS10 on the proliferation of BEL-7402 cells was analyzed by MTT method at the time of 24 h, 48 h and 72 h, respectively, and IC50 was calculated. DAPI staining was used to observe the state of BEL-7402 cells. Clone formation method was used to observe the colony formation of BEL-7402 cells, flow cytometry was used to observe the cell cycle arrest and apoptosis of BEL7402 cells, and Western blot was used to detect the expression level of PI3K/AKT pathway and related proteins. Results MTT results showed that the IC50 was(6.62±1.11)μmol·L-1; DAPI staining showed that the cell state changed significantly with the increase of drug concentration, and the results of colony formation showed that ZS10 significantly inhibited the colony formation of BEL-7402 cells. The results of flow cytometry showed that ZS10 induced S phase arrest and cycle apoptosis of BEL-7402 cells. Western blot showed that ZS10 at the concentration of 08 μ mol·L-1 could regulate the PI3K/AKT pathway and its related proteins in a dose-dependent manner. Compared with the control group, the expression of PI3K, AKT, P-AKT and anti-apoptotic protein Bcl-2 significantly decreased, the expression of pro-apoptotic protein Bax significantly increased, the expression of Cyclin D1 and CDK2 significantly decreased, and the expression of EGFR and N-cadherin, Vimentin significantly decreased in the treatment group. The expression of E-cadherin increased. Conclusions Matrine derivative ZS10 can inhibit the growth and proliferation of hepatocellular carcinoma cell line BEL-7402.

Matrine inhibits inflammatory response induced by TNF-α in human umbilical vein endothelial cells through miR-25-3p-mediated Klf4 pathway / 中国中药杂志

This study aimed to analyze the effect of matrine on tumor necrosis factor-α(TNF-α)-induced inflammatory response in human umbilical vein endothelial cells(HUVECs) and explore whether the underlying mechanism was related to the miR-25-3p-mediated Krüppel-like factor 4(Klf4) pathway. The HUVEC cell inflammation model was induced by TNF-α stimulation. After 24 or 48 hours of incubation with different concentrations of matrine(0.625, 1.25, and 2.5 mmol·L~(-1)), CCK-8 assay was used to detect cell proliferation. After treatment with 2.5 mmol·L~(-1) matrine for 48 h, the expression of TNF-α, interleukin-6(IL-6), interleukin-1β(IL-1β), and Klf4 mRNA and miR-25-3p was detected by real-time fluorescence-based quantitative PCR, and the protein expression of TNF-α, IL-6, IL-1β, and Klf4 was detected by Western blot. The anti-miR-25-3p was transfected into HUVECs, and the effect of anti-miR-25-3p on TNF-α-induced cell proliferation and inflammatory factors was detected by the above method. The cells were further transfected with miR-25-3p and incubated with matrine to detect the changes in proliferation and expression of related inflammatory factors, miR-25-3p, and Klf4. The targeting relationship between miR-25-3p and Klf4 was verified by bioinformatics analysis and dual luciferase reporter gene assay. The results displayed that matrine could inhibit TNF-α-induced HUVEC proliferation, decrease the mRNA and protein expression of TNF-α, IL-6, and IL-1β, increase the mRNA and protein expression of Klf4, and reduce the expression of miR-25-3p. Bioinformatics analysis showed that there were specific complementary binding sites between miR-25-3p and Klf4 sequences. Dual luciferase reporter gene assay confirmed that miR-25-3p negatively regulated Klf4 expression in HUVECs by targeting. The inhibition of miR-25-3p expression can reduce TNF-α-induced cell proliferation and mRNA and protein expression of TNF-α, IL-6, and IL-1β. MiR-25-3p overexpression could reverse the effect of matrine on TNF-α-induced cell proliferation and the mRNA and protein expression of TNF-α, IL-6, IL-1β, and Klf4. This study shows that matrine inhibits the inflammatory response induced by TNF-α in HUVECs through miR-25-3p-mediated Klf4 pathway.

Effects of compound matrine injection on proliferation of bladder cancer cell line BIU-87 and bladder orthotopic transplanted tumor in nude mice / 肿瘤研究与临床

Objective:To investigate the effects of compound matrine injection on the proliferation of bladder cancer cell line BIU-87 and bladder orthotopic transplantated tumor in nude mice.Methods:BIU-87 cells in logarithmic growth phase were divided into experimental group (adding 300.00, 150.00, 75.00, 37.50, 18.75 μl/ml compound matrine injection 200μl) and negative control group (adding equal volume of culturing medium). The proliferation inhibition rate and the half inhibitory concentration ( IC50) of BIU-87 cells were detected and calculated by methyl thiazole tetrazolium (MTT) method. Twenty BALB/c-nu female nude mice were injected with 100 μl of BIU-87 cell suspension with a cell density of 2×10 7/ml in the bladder to establish an animal model of bladder orthotopic transplanted tumor. After 24 hours of perfusion of BIU-87 cell suspension, intravesical perfusion administration (100 μl per nude mouse) was started, and the mice were divided into compound matrine injection group (intravesical perfusion of matrine solution) and pirarubicin group (intravesical perfusion of 1 mg/ml pirarubicin), model control group (intravesical perfusion of the same volume of sterile water), blank control group (without intravesical perfusion of BIU-87 cell suspension or administration). The observation time was 90 d. The survival status and bladder wet weight of the animals were observed and recorded, and the tumor formation rate, tumor inhibition rate and life prolongation rate were calculated. Results:Different concentrations of compound matrine injection acted on BIU-87 cells for 48 hours, and the absorbance ( A) values ??of 300.00, 150.00, 75.00, 37.50, 18.75 μl/ml compound matrine injection group and negative control group were 0.027±0.006, 0.065±0.010, 1.695±0.105, 2.387±0.017, 2.427±0.134 and 2.721±0.080 ( F = 742.67, P < 0.05), the A values ??of each concentration of compound matrine injection group were compared with the negative control group, and the differences were statistically significant (all P < 0.05). The IC50 of compound matrine injection on BIU-87 cells was 70.05 μl/ml. On the 90th day of observation, the bladder wet weights of nude mice in blank control group, model control group, pirarubicin group and compound matrine injection group were (0.018±0.004) mg, (0.422±0.130) mg, (0.219±0.136) mg and (0.237±0.113) mg ( F = 14.01, P < 0.001), and the survival time of nude mice was (90±0) d, (54±12) d, (72±4) d and (69±8) d ( F = 18.53, P < 0.001). The inhibition rates of bladder cancer in the pirarubicin group and compound matrine injection group were 48.10% and 43.84%, and the life prolongation rates of the nude mice were 34.95% and 29.53%. Conclusions:Compound matrine injection can inhibit the proliferation of BIU-87 cells in a concentration-dependent manner. Compound matrine injection can increase the tumor inhibition rate and prolong the survival time of nude mice models of bladder orthotopic transplanted tumor.

Compound Kushen injection induces immediate hypersensitivity reaction through promoting the production of platelet-activating factor via de novo pathway / 中国药理学与毒理学杂志

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 treat?ment 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 micro?vascular 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 alka?loids 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 stron?ger 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 find?ings may provide a potential approach for preventing and treating CKI-induced IHRs.

Anti-tumor and Phenotypic Regulation Effect of Matrine on Dendritic Cells through Regulating TLRs Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate the effects of matrine on antigen presentation of dendritic cells (DCs), and to explore the pharmacological mechanism of matrine on anti-tumor effect.@*METHODS@#Different concentrations (0, 1, 2, 4, 8 and 16 µ g/mL) of matrine were co-cultured with DCs, the harvested DCs were co-cultured with antigens of Lewis lung cancer (LLC) cells, and then DCs and T cells were co-cultured to produce DCs-activated killer (DAK) cells, which have significant tumor-killing activity. The expression of cytokines, mRNA and protein of toll-like receptors (TLRs) in DCs were detected by enzyme linked immunosobent assay, polymerase chain reaction and Western blot, respectively. And the killing effect of DAK were measured by MTT assay.@*RESULTS@#Matrine significantly increased the mRNA expression of TLR7, TLR8, myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF-6) and I κ B kinase (IKK), as well as the protein expression of TLR7 and TLR8, and up-regulated the levels of interleukin-12 (IL-12), IL-6 and tumor necrosis factor-α (TNF-α), meanwhile, it also increased the expressions of MHC-II, CD54, CD80 and CD86 in DCs. DCs-activated effector T cells had significant tumor-killing activity. When the concentration of matrine was more than 4 µg/mL, all indices had significant difference (P<0.01 or P<0.05).@*CONCLUSION@#Matrine plays an anti-tumor role by regulating TLRs signal transduction pathway, promoting the secretion of inflammatory cytokines and enhancing immune function.

High performance liquid chromatograohy method for determination of alkaloid, method validation in Norbu-7 granule / Монголын Анагаах Ухаан

Introduction @#The roots of Sophora Flavescentis is one of the key ingredient in Norbu 7 traditional medicine, the bioactive compound being quinolizidine alkaloids, matrine and oxymatrine. A high performance liquid chromatography (HPLC) method was used to determine matrine, oxymatrine simultaneously in the traditional medicine. The HPLC method was tested and validated for selective determination of matrine and oxymatrine in the Norbu 7 granule. The proposed method was validated for linearity, precision (system precision, method precision, intermediate or inter- day precision) and accuracy, stability in analytical solution, system suitability and ruggedness.@*Goal@#The goal of this study was to develop validated determination method of alkaloid in Norbu 7 granule for quality control.@*Material and Method@#HPLC analysis was performed on Chromecore amino bonded silica gel as the stationary phase (250 mm : 4.6 mm i.d., 5µm) using mixture of acetonitrile, dehydrated ethanol and 3% phosphoric acid (80:10:10) as the mobile phase, 220 nm as the UV light detection. </br> The research methodology was approved by Research Ethic Review Committee of Mongolian University of Pharmaceutical Science on 16th of November, 2020. @*Results@#The calibration curve of oxymatrine showed good linearity (R2=0.9955) within the established range of 8 – 64 µg/ml. The limit of detection (LOD) and quantification (LOQ) were 10.13 µg/ml and 30.71 µg/ ml respectively. Good results were achieved with repeatability (%RSD < 2.0) and recovery (93.08 – 104.32%).@*Conclusion@#The method was found to be selective, accurate, reproducible and the other components did not interfere with determinations. It was successfully used to analyze the granule traditional medicine with 7 different plant formulation and additives. The HPLC method can be used to evaluate and control quality, stability of Norbu 7 granules.

Matrine inhibits proliferation and migration of HepG2 cells by downregulating ERK1/2 signaling pathways

Objective: To research the effect of matrine on the proliferation and migration of HepG2 cells through extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. Methods: HepG2 cell was selected and divided into blank control group, experimental group (matrine 1, 2, and 4 mg/mL), and positive control group (PD98059, ERK1/2 inhibitor). MTT measure was used to detect the effective time and concentration which matrine inhibits HepG2 cells. After 24 h, the effect of effective concentration of matrine on the of morphological changing HepG2 cells was observed. The invasion ability was assayed by transwell method, the expression of ERK1/2 and pERK1/2 were detected through Western blot, and reverse transcription polymerase chain reaction was used to test the expression level of ERK1/2 mRNA. Results: With the increase of matrine concentration, the number of adherent HepG2 cells gradually decreased, the morphologic changes gradually became spherical, some cell morphology was incomplete, and even cell fragments appeared. The proliferation and invasion ability of HepG2 cells decreased. The expression of ERK1/2, pERK1/2, and ERK1/2 mRNA downregulated with the increase of matrine concentration (P < 0.05). Conclusion: Matrine inhibits the proliferation and migration of HepG2 cells by downregulating the ERK1/2 signaling pathway.

Effect of chidamide combined with matrine on proliferation and apoptosis of cutaneous T-cell lymphoma cell lines HH and Hut78 and possible apoptotic mechanisms / 中华皮肤科杂志

Objective To evaluate the effect of chidamide combined with matrine on proliferation and apoptosis of cutaneous T-cell lymphoma (CTCL) cell lines HH and Hut78,and to explore their apoptotic mechanisms.Methods Both HH and Hut78 cells were treated with 0.4 μmol/L chidamide and 0.6 g/L matrine alone or in combination for 24,48 and 72 hours,with those treated with dimethyl sulfoxide (DMSO) serving as control groups.MTS assay was performed to deteet cellular proliferation rates of HH and Hut78 cells at each time point.After 48-hour treatment,flow cytometry was conducted to detect cell apoptosis,and Western blot analysis to determine expression of apoptosis-related proteins in these cells.Statistical analysis was carried out by using repeated measures analysis of variance,one-way analysis of variance,and least significant difference (LSD)-t test for multiple comparisons.Results Compared with DMSO,chidamide and matrine alone or in combination could inhibit the proliferation of HH and Hut78cells to different extents (F =15.88,558.26,P ＜ 0.05,＜ 0.001,respectively).At 48 hours,the apoptosis rate in Hut78 cells was significantly higher in the matrine group (20.98％ ± 1.53％),chidamide group (22.44％ ± 7.74％) and combination group (44.53％ ± 1.85％) than in the control group (8.42％ ± 4.23％;LSD-t =4.76,5.31,13.69 respectively,all P ＜ 0.05),as well as in the combination group than in the matrine group and chidamide group (LSD-t =8.93,8.37 respectively,both P ＜ 0.01);no significant differences were observed in the apoptosis rate of HH cells between the matrine group (13.98％ ± 3.86％)or chidamide group (13.61％ ± 1.62％) and control group (11.44％ ± 1.43％,both P ＞ 0.05),while the combination group (20.94％ ± 0.64％) showed a significantly higher apoptosis rate compared with the control group,matrine group and chidamide group (LSD-t =7.37,5.40,5.69 respectively,all P ＜ 0.05).In the case of HH cells,the combination group showed significantly higher cleaved caspase-3 expression (all P ＜ 0.05),but significantly lower protein expression of E-cadherin,nuclear factor (NF)-κB,phosphorylated-Bad (p-Bad) and Bcl-2 compared with the other 3 groups (all P ＜ 0.05).In the case of Hut78 cells,the expression of E-cadherin,NF-κB,p-Bad and Bcl-2 was significantly lower in the matrine group,chidamide group and combination group than in the control group (all P ＜ 0.05),while cleaved caspase-3 expression was significantly higher in the chidamide group and combination group than in the control group (both P ＜0.05).No matter in HH cells or in Hut78 cells,there were no significant differences in Bad protein expression between the 4 groups (all P ＞ 0.05).Conclusion Chidamide in combination with matrine can inhibit proliferation and induce apoptosis of HH and Hut78 cells,likely by regulating the expression of apoptosis-related proteins E-cadherin,NF-κB,p-Bad,Bcl-2 and cleaved caspase-3.

Construction of Matrine Solid Lipid Nanoparticle and Determination of the Entrapment Efficiency / 中国药学杂志

OBJECTIVE: To prepare matrine solid lipoid nanoparticle,establish preparating method and determine the encapshlation efficiency. METHODS: Matrine solid lipoid nanoparticle was prepared by microemulsion-probe ultrasonic method and its quality was evaluated by particle size, Zeta potential, microscopic morphology and in vitro release. The encapsulation efficiency of the carrier was measured by different methods and their effect was compared. RESULTS: The diameter of matrine solid lipoid nanoparticle was (116.7±2.6) nm and its Zeta potential was (-45±1.7)mV. Transmission electron micrographs showed that the solid lipoid nanoparticle was uniform in size and spherical. The in vitro release result suggested the carrier exhibited control release character. Dextran gel microcolumn centrifugation can effectively separate free drugs and carriers, and the measured encapsulation efficiency data has little difference in stability. CONCLUSION: Matrine solid lipoid nanoparticle is successfully prepared and their particle size, Zeta potential and in vitro release quality are evaluated.Dextran gel microcolumn method is effective in the measurement of matrine solid lipoid nanoparticle, providing a reliable reference for the determination of water-soluble drug encapsulation efficiency.

Study on the Effects and Mechanism of Matrine on Proliferation and Collagen Synthesis of Hepatic Stellate Cells CFSC-8B Activated by Acetaldehyde / 中国药房

OBJECTIVE：To study the effects of matrine on proliferation and collagen synthesis of rat hepatic stellate cells CFSC-8B activated by acetaldehyde ，and to investigate its possible mechanism. METHODS ：CFSC-8B cells cultured in vitro were divided into blank control group ，model group ，positive control group （2.5 μmol/L colchicine）and matrine low ，medium and high concentration groups （30，60，120 μmol/L）. Except for blank control group ，other groups were activated with 200 μmol/L acetaldehyde for 24 h；medicine groups were intervened with relevant medicine for 24 h（blank control group and model group were intervened with equal volume blank medium ）. Survival rate of cell was detected by CCK- 8 assay. C ells were divided into blank control group ，model group ，positive control group （2.5 μmol/L colchicine），matrine medium and high concentration groups （60，120 μmol/L），then activated and treated with same method. Hydroxyprolin （Hyp）content in cell culture solution was tested by enzyme digestion. The contents of Col- Ⅰ and Col- Ⅲ in cell culture solution were determined by ELISA. mRNA expressionss of α-SMA，TGF-β1，TβR-І，TβR-Ⅱ，Smad3，Smad4 and Smad 7 in cells were detected by RT-PCR. The protein expressions of α-SMA，TGF-β1，TβR-Ⅰ，TβR- Ⅱ，Smad3，Smad4 and Samd 7 in cells were detected by Western blotting. RESULTS ：Compared with blank control group ，survival rate of cells in model group was increased significantly （P＜0.05）；the contents of Hyp ，Col-Ⅰ and Col- Ⅲ in cell culture solution ，mRNA and its protein expressions of α-SMA，TGF-β1，TβR-Ⅰ，TβR-Ⅱ，Smad3，Smad4 in cells were increased significantly in model group （P＜0.05），while the mRNA and protein expression of Smad 7 was decreased significantly（P＜0.05）. Compared with model group ，survival rate of cells ，the contents of Hyp ，Col-Ⅰ and Col- Ⅲ in cell culture solution，the mRNA and protein expressions of α-SMA and Smad 4 were decreased significantly in positive control group and matrine medium and high concentration groups （P＜0.05）， while the mRNA and protein expression of Smad 7 was WF-0099） increased significantly （P＜0.05）；the mRNA and proteinexpressions of TGF-β1，TβR-Ⅰ，TβR-Ⅱ and Smad 3 were decreased significantly in positive control group and matrine high concentration group （P＜0.05）. Compared with matrine medium concentration group ，all above indexes were improved significantly in matrine high concentration group （P＜0.05）. CONCLUSIONS ：Matrine can suppress the proliferation and collagen synthesis of CFSC- 8B cells activated by acetaldehyde ，with a centain concentrlation dependence ，the mechanism of which may be associated with regulating the conduction of TGFβ/Smad signal pathway.

Total alkaloids of Sophora alopecuroides- and matrine-induced reactive oxygen species impair biofilm formation of Staphylococcus epidermidis and increase bacterial susceptibility to ciprofloxacin / 中草药·英文版

Objective: To investigate the mechanism by which total alkaloids of Sophora alopecuroides (TASA) and matrine (MT) impair biofilm to increase the susceptibility of Staphylococcus epidermidis (S. epidermidis) to ciprofloxacin. Methods: The minimum biofilm inhibitory concentration (mBIC) was determined using a 2-fold dilution method. Structure of biofilm of S. epidermidis was examined by Confocal Laser Scanning Microscope (CLSM). The cellular reactive oxygen species (ROS) was determined using a DCFH-DA assay. The key factors related to the regulation of ROS were accessed using respective kits. Results: TASA and MT were more beneficial to impair biofilm of S. epidermidis than ciprofloxacin (CIP) (P < 0.05). TASA and MT were not easily developed resistance to biofilm-producing S. epidermidis. The mBIC of CIP decreased by 2–6-fold following the treatment of sub-biofilm inhibitory concentration (sub-BIC) TASA and MT, whereas the mBIC of CIP increased by 2-fold following a treatment of sub-BIC CIP from the first to sixth generations. TASA and MT can improve the production of ROS in biofilm-producing S. epidermidis. The ROS content was decreased 23%−33% following the treatment of sub-mBIC CIP, whereas ROS content increased 7%−24% following treatment with TASA + CIP and MT + CIP combination from the first to sixth generations. Nitric oxide (NO) as a ROS, which was consistent with the previously confirmed relationship between ROS and drug resistance. Related regulatory factors-superoxide dismutase (SOD) and glutathione peroxidase (GSH) could synergistically maintain the redox balance in vivo. Conclusion: TASA and MT enhanced reactive oxygen species to restore the susceptibility of S. epidermidis to ciprofloxacin.