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.

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.

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.

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.

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.

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.

Synergistic effect of a novel oxymatrine-baicalin combination against hepatitis B virus replication, a smooth muscle actin expression and typeⅠcollagen synthesis in vitro

AIM： To study the effect of oxymatrine-baicalin combination （OB） against HBV replication in 2.2.15 cells and α smooth muscle actin （ α SMA） expression, type I, collagen synthesis in HSC-T6 cells. METHODS： The 2.2.15 cells and HSC-T6 cells were cultured and treated respectively. HBsAg and HBeAg in the culture supernatants were detected by ELISA and HBV DNA levels were determined by fluorescence quantitative PCR. Total RNA was extracted from HSC-T6 cells and reverse transcribed into cDNA. The cDNAs were amplified by PCR and the quantities were expressed in proportion to β actin. The total cellular proteins extracted from HSC-T6 cells were separated by electrophoresis. Resolved proteins were electrophoretically transferred to nitrocellulose membrane. Protein bands were revealed and the quantities were corrected by β actin. RESULTS： In the 2.2.15 cell culture system, the inhibitory rate against secretion of HBsAg and HBeAg in the OB group was significantly stronger than that in the oxymatrine group （HBsAg, P = 0.043; HBeAg, P = 0.026; respectively）; HBV DNA level in the OB group was significantly lower than that in the oxymatrine group （P = 0.041）. In HSC-T6 cells the mRNA and protein expression levels of α SMA in the OB group were significantly lower as compared with those in the oxymatrine group （mRNA, P = 0.013; protein, P = 0.042; respectively）; The mRNA and protein expression levels of type I collagen in the OB group were significantly lower as compared with those in the oxymatrine group （mRNA, P 〈 0.01; protein, P 〈 0.01; respectively）.CONCLUSION： OB combination has a better effect against HBV replication in 2.2.15 cells and is more effective against α SMA expression and type I collagen synthesis in HSC-T6 cells than oxymatrine in vitro.

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.

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.

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.

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.

Treatment of pig serum-induced rat liver fibrosis with Boschniakia rossica, oxymatrine and interferon-α

AIM: To investigate the effect of Boschniakia rossica (BR),oxymatrine (OM) and interferon-alpha (IFN-α) 1b on the therapy of rat liver fibrosis and its mechanism.METHODS: By establishing a rat model of pig serum-induced liver fibrosis, liver/weight index and serum alanine transaminase (ALT) were observed to investigate the therapeutic effect of BR, OM and IFN-α. Radioimmunoassay was utilized to measure procollagen type Ⅲ (PCⅢ) and collagen type Ⅳ (CIV). RT-PCR was used to assay the expression of liver transforming growth factor- beta 1 (TGF-β1) mRNA. Immunohistochemistry of alpha-smooth muscle actin (α-SMA) and pathologic changes of liver tissues were also under investigation.RESULTS: Serum PCⅢ and CIV in BR, OM and IFN-α groups were significantly declined compared with those in model group, and their RT-PCR revealed that TGF-β1 mRNA expression was also reduced more than that in model group. Immunohistochemistry demonstrated that α-SMA also declined more than that in model group. Serum ALT in IFN-α, control and model groups was within normal level.Serum ALT in BR group had no significant difference from those of IFN-α, control and model groups. Serum ALT in OM group was significantly higher than those in BR, IFN-α,model, and control groups.CONCLUSION: BR, OM and IFN-α can prevent pig seruminduced liver rat fibrosis by inhibiting the activation of hepatic stellate cells and synthesizing collagen. OM has hepatotoxicity to rat liver fibrosis induced by pig serum.

Synergistic effect of oxymatrine and angiogenesis inhibitor NM-3 on modulating apoptosis in human gastric cancer cells

AIM: To investigate the synergistic effect of oxymatrine(OM) and angiogenesis inhibitor NM-3 on modulatingapoptosis in human gastric cancer cell lines SGC-7901,MKN-45, MKN-74. METHODS: Human gastric cancer lines SGC-7901,MKN-45, MKN-74 were treated with OM in the absenceand presence of NM-3. The inhibitory rates weredetected by MTT assay. Synergistic effect of OM andNM-3 on the growth of survivin, bcl-2, bax and p53 inSGC-7901 cells were examined by semiquantitative RT-PCR and Western blotting, and their growth inhibitoryeffects were also observed on SGC-7901 tumor xenograftin nude mice.RESULTS: OM combined with NM-3 exhibited asynergistic inhibitory effect on the growth of SGC-7901,MKN-45 and MKN-74 cells in a time-dependent manner.Twenty-four hours after treatment with OM, NM-3 aloneand their combination, mRNA expression of survivin andbcl-2 in SGC-7901 cells decreased, p53 mRNA expressionincreased. OM (4 g/L) combined with NM-3 significantlyincreased the expression of p53 mRNA and decreasedthe expression of survivin and bcl-2 compared witheither agent alone (193% ± 34% vs 129% ± 12%;44% ± 18% vs 92% ± 18%; 36 ± 17% vs 93% ± 23%,P < 0.05). Western blotting showed that the synergisticeffect of OM and NM-3 on protein translation of survivin,bcl-2 and p 53 was in accordance with their mRNAs.Furthermore, OM/NM-3 combination obviously exhibitedantitumor growth effect in xenografted human gastriccancer cells SGC-7901 compared with either agent alone.CONCLUSION: OM combined with NM-3 has synergisticinhibitory effects on human gastric cancer cells in vitro and can suppress the growth of xenografted human gastric cancer cells SGC-7901 in vivo.

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.

Oxymatrine Improves TNBS-induced Colitis in Rats by Inhibiting the Expression of NF-κB p65

Inflammatory bowel disease is thought to be regulated by the balance between Th1 and Th2 cytokines secreted by T cells, and NF-κB p65 also plays a predominant role in the intestinal inflammation. We evaluated the potency of oxymatrine, one of active components of Sophora Root, in inhibiting the immune responses and inflammation in 2,4,6-trinitrobenzene sulfonic acid （TNBS）-induced colitis. The inflammation was markedly ameliorated in the oxymatrine-treated rats. The level of IL-2 was increased and that of IL-10 was decreased in colon tissue in the rat model, which was reversed by the treatment of oxymatrine. Moreover, the elevated expression of NF-κB p65 in colon tissue in the model was also improved by oxymatrine treatment. Our results suggest that oxymatrine might be beneficial for the abnormal immune responses and inflammation by regulating the unbalance of Th1 and Th2 cytokines secretion and inhibiting the expression of NF-κB p65 in colon tissue.

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.

Effect of oxymatrine on interferon-gamma and tumor necrosis factor-alpha serum levels in an experimental rat model of autoimmune encephalomyelitis

BACKGROUND： Studies have demonstrated that experimental autoimmune encephalomyelitis （EAE） onset correlates with increased interferon-v （IFN-γ） and tumor necrosis factor-α （TNF-α） expression. Oxymatrine （OM） has been shown to inhibit autoimmune responses, but there are no reports showing that it could prevent the development of EAE. OBJECTIVE： To observe the effect of OM on serum levels of IFN-γ and TNF-α in a rat model of EAE.DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Experimental Animal Center of Henan Academy of Chinese Medicine and at the Key Disciplines Laboratory Clinical Medicine of Henan Province between July and December 2008. MATERIALS： OM was purchased from Chia-tai Tianqing Pharmaceutical, China; complete Freund＇s adjuvant was purchased from Sigma, USA. METHODS： Forty female Wistar rats were randomly assigned to four groups： EAE model （M）, low-dose OM treatment （OM-L）, high-dose OM treatment （OM-H）, and normal control （N, no immunization）, with 10 rats in each group. EAE was established in the M, OM-L, and OM-H groups following immunization with Guinea pig spinal cord homogenate and complete Freund＇s adjuvant. The M and N groups were intraperitoneally injected with normal saline （6.7 mL/kg per day）, the OM-L group received an intraperitoneal injection of OM （100 mg/kg per day）, and the OM-H group received OM （150 mg/kg per day）. MAIN OUTCOME MEASURES： At 16 days after immunization, the degree of histopathological changes in the spinal cord was assessed by hematoxylin-eosin stanining. Enzyme-linked immunosorbent assay was used to detect serum levels of IFN-γ, and radioimmunoassay was utilized to determine serum TNF-α level. Neurological scores were measured on a daily basis according to a 0-5 scale. RESULTS： Daily injections of OM, both high and low doses, resulted in decreased neurological scores in EAE rats （P〈0.01）, as well as reduced cellular infiltration in the spinal cord and decreased levels of serum IFN-γ and TNF-α （P〈 0.01）. CONCLUSION： OM reduced the onset and severity of EAE, which correlated with decreased IFN-γ and TNF-α expression.

Synthesis of N-Succinyl-chitosan(Suc-Chi) and Preparation of Oxymatrine(OM)/N-Succinyl-chitosannanoparticles

Oxymatrine （OM）/N-succinyl-chitosan （Suc-Chi, with a degree of substitution being 0. 32） was synthesized via the ring-opening reaction of succinic anhydride with chitosan in dimethyl sulfoxide. OM-loaded Suc-Chi nanoparticles were prepared by an ionotropic gelation process and OM was quantified via the HPLC method： The influences of the initial OM concentration on the nanoparticle characteristics and OM release behavior were evaluated. The nanoparticles were found to have a mean diameter within a range of 267-392 nm, a positive surface charge, and a zeta potential in the range of 19-27 inV. The formulation with an initial OM concentration of 100μg/mL provided the highest loaded capacity（0. 77% ） and the highest extent of the released OM （68% at 24 h）, suggesting the possibility to achieve a therapeutic dose. According to the data obtained, this Suc-Chi-based nanotechnology will open up new and interesting prospects for the development of new anticancer drugs.

Effects of Oxymatrine on the NF-kappa B expression of HaCaT cells

Objective： To study the effect of oxymatrine on the expression of nuclear factor-kappa B（NF-K B） of Human benign epidermal keratinocytes line（HaCaT cells）. Methods：HaCaT cells were cultured with different concentration of Oxymatrine（10 μ g/ml, 50 μ g/ ml and 100 μg/ml） for 24 h, then 10.5 mol/L Substance P was added to the cells. After 30 min, NF-K B expression in the cells was observed by immunocytochemistry, NF-K B P65 protein expression was evaluated by Western blot, and the mRNA expression of NF-K B P65 was evaluated by reverse transcription polymerase chain reaction（RT-PCR）. The 10.5 mol/L Substance P and culture medium were added to the Substance P group and control group, respectively. Results：In control group, expression rate of positive cells, the expressions of protein and mRNA of NF-K B were all low. In Substance P group, when 10μ mol/L Substance P was added, the expressions were all increased（P 〈 0.05）. But in Oxymatrine groups, the expression rate of positive cells, the expressions of protein and mRNA were all descended in a concentration-dependent manner（P 〈 0.05 or P 〈 0.01）. Conclusion：Oxymatrine can down-regulate the expression of NF-K B of the HaCaT cells and may play an important role in regulating anti-inflammation and immunity.