Network pharmacology-based exploration of pathways involved in the inhibition of hepatocellular carcinoma cell proliferation by Oxymatrine

Objective:To explore the effect of Oxymatrine(OMT)on hepatocellular carcinoma cells and its possible action pathways.Methods:The core targets of Oxymatrine action in hepatocellular carcinoma were identified using network pharmacology and validated using in vitro experiments.CCK8 assay and scratch assay were used to detect the effects of Oxymatrine on the proliferation and migration of MHCC97H(TP53 mutant)cells,and Western blot assay and qPcr assay were used to find out the expression of the core targets after Oxymatrine acted on MHCC97H cells at the protein level and mRNA level,respectively.Results:Network pharmacology showed that the core target was TP53 protein;CCK8 assay showed that the IC50 at 24 h and 48 h was 9.64 mg/ml and 7.47 mg/ml,respectively;Scratch assay proved that Oxymatrine markedly inhibited the migratory ability of MHCC97H cells;Western blot assay and qPcr assay found that TP53 gene expression was down-regulated.The western blot experiment and qPcr experiment found that TP53 gene expression was down-regulated.Conclusions:Oxymatrine significantly inhibited the proliferation and migration of hepatocellular carcinoma MHCC97H cells,and its mechanism of action may be related to TP53.

Chemical derivatization strategies for enhancing the HPLC analytical performance of natural active triterpenoids

Triterpenoids widely exist in nature,displaying a variety of pharmacological activities.Determining triterpenoids in different matrices,especially in biological samples holds great significance.High-performance liquid chromatography(HPLC)has become the predominant method for triterpenoids analysis due to its exceptional analytical performance.However,due to the structural similarities among botanical samples,achieving effective separation of each triterpenoid proves challenging,necessitating significant improvements in analytical methods.Additionally,triterpenoids are characterized by a lack of ultraviolet(UV)absorption groups and chromophores,along with low ionization efficiency in mass spectrometry.Consequently,routine HPLC analysis suffers from poor sensitivity.Chemical derivatization emerges as an indispensable technique in HPLC analysis to enhance its performance.Considering the structural characteristics of triterpenoids,various derivatization reagents such as acid chlorides,rhodamines,isocyanates,sulfonic esters,and amines have been employed for the derivatization analysis of triterpenoids.This review comprehensively summarized the research progress made in derivatization strategies for HPLC detection of triterpenoids.Moreover,the limitations and challenges encountered in previous studies are discussed,and future research directions are proposed to develop more effective derivatization methods.

Simultaneous determination of five plant hormones in cotton leaves using QuEChERS combined with HPLC‒MS/MS

Background Plant hormones profoundly influence cotton growth,development,and responses to various stresses.Therefore,there is a pressing need for an efficient assay to quantify these hormones in cotton.In this groundbreaking study,we have established QuEChERS-HPLC‒MS/MS method,for the simultaneous detection of multiple plant hormones in cotton leaves,allowing the analysis and quantification of five key plant hormones.Results Sample extraction and purification employed 0.1%acetic acid in methanol and C18 for optimal recovery of plant hormones.The method applied to cotton demonstrated excellent linearity across a concentration range of 0.05–1 mg・L−1,with linear regression coefficients exceeding 0.99.The limits of quantification(LOQs)were 20μg・kg−1 for GA3 and 5μg・kg−1 for the other four plant hormones.Recovery rates for the five plant hormones matrix spiked at levels of 5,10,100,and 1000μg・kg−1 were in the range of 79.07%to 98.97%,with intraday relative standard deviations(RSDs)ranging from 2.11%to 8.47%.The method was successfully employed to analyze and quantify the five analytes in cotton leaves treated with plant growth regulators.Conclusion The study demonstrates that the method is well-suited for the determination of five plant hormones in cotton.It exhibits excellent selectivity and sensitivity in detecting field samples,thus serving as a robust tool for indepth research into cotton physiology.

Rapid Analysis of Four Alkaloids in Uncaria rhynchophylla by Core-Shell Column HPLC and Quantitative Analysis of Multi-Components by Single Marker(QAMS)

As a traditional herbal medicine,the major alkaloids in Uncaria rhynchophylla have been proven to have blood pressure-lowering and sedative effects.It is essential to develop an effective method for the determination of the major alkaloids in U.rhynchophylla.In this research,a rapid quantitative analysis involving multi-components analysis by a single marker strategy coupled with core-shell column HPLC was adopted to analyse four alkaloids(corynoxeine,isocorynoxeine,isorhynchophylline,rhynchophylline)in U.rhynchophylla.Isorhynchophylline was selected as the internal reference substance,the content of which was determined by the traditional external standard method.Relative correction factors(RCF)between isorhynchophylline and the other three alkaloids were calculated respectively.The results showed that the QAMS method had good robustness under different HPLC instruments.Nineteen batches of U.rhynchophylla were tested.No significant difference was observed between the results by QAMS and EMS(Correlation coefficient>0.99,p>0.05).The QAMS method could be employed as a rapid,effective technique for the quality control of U.rhynchophylla.

Preparation and study of anti-hepatitis B virus activity in vitro of oxymatrine phospholipid complex

Aim The oxymatrine phospholipid complexs were prepared, and its activity against hepatitis B virus in vitro were studied. Methods Using tetrahydrofuran as a reaction medium, oxymatrine and phospholipids were resolved into the medium, Oxymatrine phospholipid eomplexs were prepared. The toxicity measurements and inhibitory effect on HBsAg, HBeAg, and HBVDNA of oxymatrine phospholipid complex in 2.2.15 cells were studied respectively. Results The content of oxymatrine in the phospholipids eomplexs prepared was 24,86% （W/W）. The TCO of the oxymatrine phospholipid eomplexs was 250 μmol·L^- 1 The inhibitory effect of HBsAg, HBeAg, HBV-DNA of 2.2.15 cells treated by oxymatrine phospholipid complex were higher than those of the oxymatrine. Conclusion Oxymatrine phospholipid complex can have stronger effective activity against hepatitis B virus compared with oxymatrine. So oxymatrine phospholipid eomplexs were showing its potential antiviral activity in hepatitis B treatment.

Evaluation of Uncertainty for Determination of Stevioside Content in Fermented Milk by HPLC

[Objectives]The paper was to establish an evaluation method for the uncertainty of stevioside(including stevioside,rebaudioside A,rebaudioside B,rebaudioside C,rebaudioside F,Dulcoside A,rubusoside and steviolbioside)content determination in fermented milk based on HPLC.[Methods]The mathematical model of stevioside content and the propagation rate of uncertainty were established,and the sources of uncertainty were analyzed.[Results]The uncertainty mainly came from four main aspects,including standard uncertainty u(C)introduced by solution concentration C,standard uncertainty u(V)introduced by sample volume V,standard uncertainty u(m)introduced by sample mass m weighing and standard uncertainty u(f_(rep))introduced by measurement repeatability of stevioside content after sample dissolution and constant volume.The uncertainty estimation table and fishbone chart of stevioside content X determination were established.The relative synthetic standard uncertainty of stevioside content was obtained,and the standard uncertainty was extended to form the measurement result of stevioside content and its uncertainty report.[Conclusions]The evaluation results can be directly applied to the daily practical detection work.

Thermodynamics Properties of Oxymatrine in NaCl Solution

The enthalpies of dissolution of oxymatrine in 0.9%NaCl solution were measured using a RD496-2000 Calvet Microcalorimeter at 309.65 K under atmospheric pressure. The differen tial enthalpy and molar enthalpy of oxymatrine dissolution in the 0.9%NaCl solution of were determined. The corresponding kinetic equation that described the dissolution process was elucidated. Moreover, the half-life, molar entropy, molar enthaply, and Gibbs free energy of the dissolution process were also obtained.

Validation of a Method for the Measurement of Caffeine in Water by HPLC-UV

For the production of a reference material from caffeine solution, one of the methods of characterization was HPLC-UV since caffeine is very sensitive to the UV. In this work, a batch solution of caffeine in water reference material of 1000 mg/kg has been gravimetrically prepared using a calibrated analytical balance. A sample of this solution was diluted to 25 mg/kg for measurement by HPLC-UV in the range 10 - 50 mg/kg. The chromatographic separation was carried out by C-18 column and a mobile phase assembled of 75% water and 25% methanol (v:v). The detection was made by the UV detector at 275 nm. The validation of this analytical method was carried out in accordance with requirements of the EURACHEM and ICH guidelines. The selectivity, linearity, accuracy, precision and trueness (recovery and bias) of the method were studied. The validation results proved that the method is fit-for-purpose of measuring the caffeine concentration in water in the range 10 - 50 mg/kg using HPLC-UV.

Silver-ion HPLC测定1,3-二油酸-2-棕榈酸三酰甘油的含量

准确测定1,3-二油酸-2-棕榈酸三酰甘油(OPO)的含量,建立了一种以高效液相色谱结合银离子交换柱(Silver-ion HPLC)的测定方法。以ChromSpher 5 Lipid(25 cm×4.6 mm,5μm)银离子交换柱通过HPLC正相系统分离三酰甘油,配合蒸发光散射检测器(ELSD),以毒性较小的二氯甲烷和丙酮为流动相。流速0.6 mL/min,柱温30℃。漂移管温度30℃,空气压力0.35 MPa。在此条件下,1,3-二油酸-2-棕榈酸三酰甘油和1,2-二油酸-3-棕榈酸三酰甘油两种同分异构体基本达到分离,样品出峰完整,峰形良好。OPO色谱峰面积的自然对数与浓度的自然对数呈良好的线性关系(R2=0.998 8)。用该方法测定了母乳脂肪、实验室合成结构油脂样品、商品结构油脂、棕榈油和山茶油中OPO的含量。结果表明该方法的灵敏度高、重现性良好。

Effect of oxymatrine on the replication cycle of hepatitis B virus in vitro

AIM:To determine the antiviral mechanism or target of oxymatrine against hepatitis B virus(HBV).METHODS:HepG2.2.15 cells were incubated with culture medium containing 500 μg/mL of oxymatrine for 2 and 5 d.The surface antigen of HBV(HBsAg) and e antigen of HBV(HBeAg) in supernatant were determined by ELISA.HBV DNA in supernatant,and intracellular covalently closed circular DNA(cccDNA),relaxed circular DNA(rcDNA) and pregenomic RNA(pgRNA) were quantif ied by specif ic real-time polymerase chain reaction(PCR) or reverse transcription(RT)-PCR.RESULTS:Treatment with oxymatrine for 2 d and 5 d reduced the production of HBV by the cell line,as indicated by the decline of HBsAg(22.67%,t = 5.439,P = 0.0322 and 22.39%,t = 5.376,P = 0.0329,respectively),HBeAg(55.34%,t = 9.859,P = 0.0101 and 43.97%,t = 14.080,P = 0.0050) and HBV DNA(40.75%,t = 4.570,P = 0.0447 and 75.32%,t = 14.460,P = 0.0047) in the supernatant.Intracellular cccDNA was also markedly reduced by 63.98%(t = 6.152,P = 0.0254) and 80.83%(t = 10.270,P = 0.0093),and intracellular rcDNA by 34.35%(t = 4.776,P = 0.0413) and 39.24%(t = 10.050,P = 0.0097).In contrast,intracellular pgRNA increased by 6.90-fold(t = 8.941,P = 0.0123) and 3.18-fold(t = 7.432,P = 0.0176) after 500 μg/mL of oxymatrine treatment for 2 d and 5 d,respectively.CONCLUSION:Oxymatrine may inhibit the replication of HBV by interfering with the process of packaging pgRNA into the nucleocapsid,or inhibiting the activity of the viral DNA polymerase.

Role of DOR-β-arrestin1-Bcl2 Signal Transduction Pathway and Intervention Effects of Oxymatrine in Ulcerative Colitis

This study was aimed to investigate the role of the delta-opioid receptor （DOR）-β-arrestinl-Bcl-2 signal transduction pathway in the pathogenesis of ulcerative colitis （UC） and the intervention effects of oxymatrine on UC. Forty Sprague-Dawley rats were divided into nor- mal group, model group, oxymatrine-treated group and mesalazine-treated group （n=10 each） at ran- dom. The rat UC model was established by intra-colonic injection of trinitrobenzene sulfonic acid in the model group and two treatment groups. The rats in oxymatrine-treated group were subjected to intramuscular injection of oxymatrine [63 mg/（kg·day）] for 15 days, and those in mesalazine-treated group given mesalazine solution [0.5 g/（kg·day）] by gastric lavage for the same days. Animals in normal group and model group were administered 3 mL water by gastric lavage for 15 days. On the 16th day, after fasting for 24 h, the rats were sacrificed for the removal of colon tissues. The expres- sion levels of DOR, β-arrestinl and Bcl-2 were determined in colon tissues by immunohistochemistry and real-time quantitative polymerase chain reaction （RT-PCR）, respectively. It was found that the expression levels of DOR, [3-arrestinl and Bcl-2 protein and mRNA were significantly increased in the model group as compared with the other groups （P〈0.05）. They were conspicuously decreased in both mesalazine-treated and oxymatrine-treated groups in contrast to the model group （P〈0.05）. No statistically significant difference was noted in these indices between mesalazine- and oxyma- trine-treated groups （P〉0.05）. This study indicated that the DOR-β-arrestinl-Bcl-2 signal transduc- tion pathway may participate in the pathogenesis of UC. Moreover, oxymatrine can attenuate the de- velopment of UC by regulating the DOR-β-arrestin 1-Bcl-2 signal transduction pathway.

Inhibitory effect of oxymatrine on hepatocyte apoptosis via TLR4/PI3K/Akt/GSK-3β signaling pathway

To evaluate the effect of oxymatrine (OMT) on hepatocyte apoptosis in rats with lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced acute liver failure (ALF).METHODSLPS/D-GalN was used to establish a model of ALF in rats. To evaluate the effect of OMT, we assessed apoptosis by transmission electron microscopy, and the pathological changes in the liver by light microscopy with hematoxylin and eosin staining. An automated biochemical analyzer was used to measure serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Enzyme-linked immunosorbent assay was used to determine the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β. Western blotting was used to detect protein levels in liver tissues. Streptavidin peroxidase immunohistochemistry was used to observe expression of Toll-like receptor (TLR)4, active caspase-3, Bax and Bcl-2.RESULTSAll rats in the normal control and OMT-pretreated groups survived. The mortality rate in the model group was 30%. OMT preconditioning down-regulated apoptosis of hepatocytes and ameliorated pathological changes in liver tissue. The levels of AST, ALT, TNF-α and IL-1β in the model group increased significantly, and were significantly reduced by OMT pretreatment. OMT pretreatment down-regulated expression of TLR4 and active caspase-3 and the Bax/Bcl-2 ratio, and up-regulated expression of P-AktSer473 (Akt phosphorylated at serine 473) and P-GSK3βSer9 (glycogen synthase kinase 3β phosphorylated at serine 9) induced by LPS/D-GalN.CONCLUSIONOMT inhibits hepatocyte apoptosis by suppressing the TLR4/PI3K/Akt/GSK-3β signaling pathway, which suggests that OMT is an effective candidate for ameliorating acute liver failure.

A rapid HPLC method for determination of coniferyl ferulate in Angelica sinensis and Ligusticum chuanxiong

Aim A reliable and rapid HPLC method was developed for quantitative determination of coniferyl femlate, an ester of ferulic acid, with multiple pharmacological activities in Angelica sinensis and Ligusticum chuanxiong, two commonly used Chinese medicines. Methods The determination was achieved by using a Zorbax ODS C18 analytical column （250 mm×4.6 mm ID, 5 μm） at isocratic elution of 1% aqueous acetic acid and acetonitrile （1：1） with diode-array detection （318 nm）. The calibration curve of coniferyl femlate showed good linearity （r^2 = 0.9995） within the test range. Results The developed method showed good precision with intra- and inter-day variations of 0.22% - 1.16% and 0.86% - 2.62% between the levels of 0.380 - 0.038 mg·mL^-1, respectively. The repeatability represented as RSD of coniferyl femlate was less than 2.7% for three levels （0.2 - 1.0 g of Angelica sinensis）, and the recovery was 105.3% with RSD of 3.2%. Conclusion The validated method was successfully applied to quantify coniferyl femlate in 12 samples of Danggui and Chuanxiong.

Effect of Oxymatrine on the TGFbeta-Smad signaling pathway in rats with CCl_4-induced hepatic fibrosis

AIM： To explore the anti-fibrotic effect of Oxymatrine on CCl4-induced liver fibrosis in rats and its modulation on the TGFbeta-Smad signaling pathway. METHODS： One hundred healthy male SD rats were randomly divided into three groups： normal group （n = 20）, treatment group of Oxymatrine （n = 40） and CCh-induced fibrosis group （n = 40）. Experimental hepatic fibrosis was induced by subcutaneous injection of carbon tetrachloride （CCh soluted in liquid paraffin with the concentration of 300 g/L, the dosage of injection was 3 mL/kg, twice per week for 8 wk）. The treated rats received Oxymatrine via celiac injection at a dosage of 10 mg/kg twice a week at the same time. The deposition of collagen was observed with H＆E and Masson staining. The concentration of serum TGF-β1 was assayed with ELISA. The gene expression of Smads and CBP （CREB binding protein） was detected with in situ hybridization （ISH） and immunohistochemistry （IH）, respectively. All the experimental figures were scanned and analyzed with special figure-analysis software. RESULTS： A significant reduction of collagen deposition and rearrangement of the parenchyma was noted in the liver tissue of Oxymatrine-treated rats. The semi- quantitative histological scores （2.43 ± 0.47 μm^2 vs 3.76 ±0.68, P 〈 0.05） and average area of collagen/in those rats were significantly decreased when compared with hepatic cirrhosis model rats （94.41 ± 37.26 μm^2 vs 290.86 ± 89.37 μm^2, P 〈 0.05）. The gene expression of Smad 3 mRNA was considerably decreased in the treated animals. The A value of Smad 3 mRNA was lower in the treated rats than the model rats （0.034 ± 0.090 vs 0.167 ± 0.092, P 〈 0.05）. Contrarily, the A value of Smad 7 mRNA was increased considerably in the treated animals （0.175 ± 0.065 vs 0.074 vs 0.012, P 〈 0.05）. There was an obvious decrease in the expression of CBP mRNA in treated rats as illuminated by a reduction of its A value when compared with model rats （0.065±0.049 vs 0.235 ± 0.025, P 〈 0.001）. CONCLUSION： Oxymatrine is effective in reducing the production and deposition of collagen in the liver tissue of experimental rats. Oxymatrine could promote the expression of Smad 7 and inhibit the expression of Smad 3 and CBP in CCh-induced hepatic fibrosis in SD rats, could modulate the fibrogenic signal transduction of TGFβ-Smad pathway.

Effects of Oxymatrine on the Apoptosis of Human Esophageal Carcinoma Eca109 Cell Line and Its Mechanism

The effects of Oxymatrine （Oxy） on the proliferation and apoptosis of human esophageal carcinoma Ecal09 cell line and the mechanism were investigated. The human esophageal carcinoma Eca 109 celis were cultured in vitro. The Oxy-induced apoptosis of Eca 109 cells was assayed by using flow cytometry. The expressions of p-ERKII2, Cyclin D1, p21^waf/cipl, Bax and Bcl-2 were detected by Western blot. Flow cytometry revealed that Oxy could induce the apoptosis of Eca l09 cells. Western blot showed that Oxy of different concentrations suppressed the expressions of p-ERK1/2, Cyclin D1 and Bcl-2, but up-regulated the expression of p21waf/cip1 and Bax, and the ratio of Bax/Bcl-2 was increased. It was suggested the Oxy could induce the apoptosis of Eca l09 cells, which might be related to the upregulation of p21waf/cip1 and the downregulation of p-ERK1/2, Cyclin D1 and p21^waf/cip1. The possible pathway may be related to Bcl-2/Bax.

Anti-inflammatory mechanism of oxymatrine in dextran sulfate sodium-induced colitis of rats

AIM： To investigate the anti-inflammatory mechanism of oxymatrine in dextran sulfate sodium （DSS）-induced colitis of rats. METHODS： Acute colitis was induced by giving 2% DSS orally in drinking water for 8 d. Twenty-six male rats were randomized into oxymatrine-treated group （group A, 10 rats）, DSS control （group B, 10 rats） and normal control （group C, 6 rats）. The rats in group A were injected muscularly with oxymatrine at the dosage of 63 mg/（kg·d） from d 1 to 11 and drank 2% DSS solution from d 4 to 11. The rats in group B were treated with 0.9% saline in an equal volume as group A and drank 2% DSS solution from d 4 to 11. The rats in group C were treated with 0.9% saline as group B from d 1 to 11 and drank water normally. Diarrhea and bloody stool as well as colonic histology were observed. The levels of serum tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） were determined by ELISA, and nuclear factor-κB （NF-κB） activity and the expression of inter-cellular adhesion molecule-1 （ICAM-1） in colonic mucosa were detected by immunohistochemistry method. RESULTS： Compared with DSS control group, the inflammatory symptoms and histological damages of colonic mucosa in oxymatrine-treated group were significantly improved, the serum levels of TNF-α, IL-6, and the expression of NF-κB, ICAM-1 in colonic mucosa were significantly reduced. CONCLUSION： The fact that oxymatrine can reduce the serum levels of TNF-α, IL-6, and the expression of NF-κB and ICAM-1 in colonic mucosa in DSS-induced colitis of rats indicates that oxymatrine may ameliorate the colonic inflammation and thus alleviate diarrhea and bloody stool.

Analysis of Monosaccharide Composition of Pu-erh Tea Polysaccaride by Pre-column Derivatization HPLC

[Objective] This study aimed to establish a pre-column derivatization HPLC method for the identification and analysis of monosaccharide composition of Pu-erh tea polysaccharide. [Method] Pu-erh tea polysaccharide was extracted using the wa- ter extraction method, further isolated and purified by DEAE cellulose-52 columns. The obtained tea polysaccharide and four components TPS1, TPS2, TPS3 and TPS, were first derived by 1-phenyl-3-methyl-5-pyrazolone （PMP）, and then the PMP derivatives of monosaccharide were analyzed by high performance liquid chromatog- raphy （HPLC）. [Result] Pu-erh tea polysaccharide contained eight kinds of monosac- chaddes （mannose, rhamnose, glucuronic acid, galacturonic acid, grucose, galactose, arabinose, fucose）, without xylose; so it was the same with TPS1; each of TPS2, TPS3 and TPS4 contained seven monosaccharides, while no fucose. [Conclusion] This method is simplified and rapid, which can be used to determine the monosac- charide composition of Pu-erh tea polysaccharide and monosaccharide content.

Oxymatrine reduces neuroinflammation in rat brain A signaling pathway

Cerebral neuroinflammation models were established by injecting 10μg lipopolysaccharide into the hippocampus of male Sprague-Dawley rats. The rats were treated with an intraperitoneal injection of 120, 90, or 60 mg/kg oxymatrine daily for three days prior to the lipopolysaccharide injection. Twenty-four hours after model induction, the hippocampus was analyzed by real-time quantitative PCR, and the cerebral cortex was analyzed by enzyme-linked immunosorbent assay and western blot assay. The results of the enzyme-linked immunosorbent assay and the real-time quantitative PCR showed that the secretion and mRNA expression of the pro-inflammatory cytokines interleukin-113 and tumor necrosis factor-a were significantly decreased in the hippocampus and cerebral cortex of model rats treated with oxymatrine. Western blot assay and real-time quantitative PCR analysis indicated that toll-like receptor 4 mRNA and protein expression were significantly decreased in the groups receiving different doses of oxymatrine. Additionally, 120 and 90 mg/kg oxymatrine were shown to reduce protein levels of nuclear factor-KB p65 in the nucleus and of phosphorylated IKBa in the cytoplasm of brain cells, as detected by western blot assay. Experimental findings indicate that oxymatrine may inhibit neuroinflammation in rat brain via downregulating the expression of molecules in the toll-like receptor 4/nuclear factor-KB signaling Dathwav.

Effects of oxymatrine on experimental hepatic fibrosis and its mechanism in vivo

AIM: Hepatic fibrogenesis has close relation with hepatic stellate cells (HSC)and tissue inhibitors of metalloproteinase (TIMP). Oxymatrine (OM) is a kind of Chinese herb that is found to have some effects on liver fibrosis. We aimed to determine the effects of OM on hepatic fibrosis and explore the possible mechanism. METHODS: Thirty-two rats were randomly divided into four groups; 16 were used to develop hepatic fibrosis by carbon tetrachloride (CCI4) and treated with or without OM, and 16 were used as controls. The expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and α-smooth muscle actin (α-SMA) in the livers of rats was detected by immunohisto-chemical assay. Liver pathology was determined by H&E staining and reticulum staining. RESULTS: In CCl4-injured rats, the normal structure of lobules was destroyed, and pseudolobules were formed. Hyperplasia of fibers was observed surrounding the lobules. While the degree of fibrogenesis in liver tissues was significantly decreased in those rats with OM-treatment compared with those without OM treatment. The pseudolobules were surrounded by strong, multi-layer reticular fibers, which netted into pseudolobules in CCl4-injured rats, however, there was a significant decrease in reticular fibers in OM-treated rats. The expression of TIMP-1 in hepatic cells was weak in control groups, but strong in CCl4-injured groups, however, the expression of TIMP-1 was significantly inhibited by OM (F = 52.93, P<0.05). There was no significant change in the expression of α-SMA between CCl4-injured rats with or without OM treatment (F= 8.99, P>0.05). CONCLUSION: OM effectively inhibits CCl4-induced fibrogenesis in rat liver tissues, probably by reducing the expression level of TIMP-1.

Oxymatrine liposome attenuates hepatic fibrosis via targeting hepatic stellate cells

AIM： To investigate the potential mechanism of Arg- Gly-Asp （RGD） peptide-labeled liposome loading oxy- matrine （OM） therapy in CCI4-induced hepatic fibrosis in rats. METHODS： We constructed a rat model of CCh- induced hepatic fibrosis and treated the rats with dif- ferent formulations of OM. To evaluate the antifibrotic effect of OM, we detected levels of alkaline phospha- tase, hepatic histopathology （hematoxylin and eosin stain and Masson staining） and fibrosis-related gene expression of matrix metallopeptidase （MMP）-2, tis- sue inhibitor of metalloproteinase （TIMP）-I as well as type I procollagen via quantitative real-time poly- merase chain reaction. To detect cell viability and apop- tosis of hepatic stellate cells （HSCs）, we performed 3-（4,5）-dimethylthiahiazo（-z-yl）-3,5-diphenytetrazoli- umromide assay and flow cytometry. To reinforce the combination of oxymatrine with HSCs, we constructed fluorescein-isothiocyanate-conjugated Arg-Gly-Asp peptide-labeled liposomes loading OM, and its targeting of HSCs was examined by fluorescent microscopy. RESULTS： OM attenuated CCh-induced hepatic fibro- sis, as defined by reducing serum alkaline phosphatase （344.47± 27.52 U/L vs 550.69 ± 43.78 U/L, P 〈 0.05）, attenuating liver injury and improving collagen deposits （2.36% ± 0.09% vs 7.70% ±0.60%, P 〈 0.05） and downregulating fibrosis-related gene expression, that is, MMP-2, TIMP-1 and type I procollagen （P 〈 0.05）. OM inhibited cell viability and induced apoptosis of HSCs in vitro. RGD promoted OM targeting of HSCs and en- hanced the therapeutic effect of OM in terms of serum alkaline phosphatase （272.51 ± 19.55 U/L vs 344.47 ± 27.52 U/L, P 〈 0.05）, liver injury, collagen deposits （0.26%± 0.09% vs 2.36% ± 0.09%, P 〈 0.05） and downregulating fibrosis-related gene expression, that is, MMP-2, TIMP-1 and type I procollagen （P 〈 0.05）. Moreover, in vitro assay demonstrated that RGD en- hanced the effect of OM on HSC viability and apoptosis. CONCLUSION： OM attenuated hepatic fibrosis by in- hibiting viability and inducing apoptosis of HSCs. The RGD-labeled formulation enhanced the targeting effi- ciency for HSCs and the therapeutic effect.