[The effects of Shadu Cao Mixture on immune functions of immunosuppression mice].

OBJECTIVE: To investigate the effects of Shadu Cao Mixture (SDCM, traditional Chinese medicine) on immune functions of immunosuppression mice. METHODS: Fifty BALB/C mice were randomly divided into blank control group, model group, SDCM low-dose, middle-dose and high-dose group. Except the blank control group, other groups were intraperitoneal injected with cyclophosphamide (40 mg/kg) to establish immunosuppression mice model. The blank control group and model group received gavage administration with nonnal saline, while the other groups received gavage administration with different doses of SDCM (10, 20, 40 m/kg for 15 days) respectively. The number of leukocytes and serum levels of interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in peripheral blood, spleen index, and the function of NK cells were measured. RESULTS: Compared with the model group , SDCM increased the number of leukocytes and serum concentrations of IL-2, TNF-α and IFN-γ in peripheral blood and improved the spleen index and the function of NK cells significantly (P < 0.05-0.01). CONCLUSION: SDCM could remarkably enhance the immune functions of immunosuppression mice induced by cyclophosphamide.

5,7-Dihydroxyflavone Enhances the Apoptosis-Inducing Potential of TRAIL in Human Tumor Cells via Regulation of Apoptosis-Related Proteins.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for the treatment of cancer, because it preferentially induces apoptosis in numerous cancer cells with little or no effect on normal cells. 5,7-Dihydroxyflavone is a dietary flavonoid commonly found in many plants. Here we show that the combined treatment with 5,7-dihydroxyflavone and TRAIL at subtoxic concentrations induced strong apoptotic response in human hepatocarcinoma HepG2 cells, acute leukemia Jurkat T cells, and cervical carcinoma HeLa cells. We further investigated the mechanisms by which 5,7-dihydroxyflavone augments TRAIL-induced apoptosis in HepG2 cells. 5,7-Dihydroxyflavone up-regulated the expression of pro-apoptotic protein Bax, attenuated the expression of anti-apoptotic proteins Bcl-2, Mcl-1, and IAPs, and reduced the phosphorylation levels of Akt and STAT3, weakening the anti-apoptotic signals thus facilitating the process of apoptosis. Moreover, 5,7-dihydroxyflavone and TRAIL were well tolerated in mice, and the combination of 5,7-dihydroxyflavone and TRAIL reduced tumor burden in vivo in a HepG2 tumor xenograft model. Interestingly, 5,7-dihydroxyflavone-mediated sensitization to TRAIL-induced cell death was not observed in normal human hepatocytes L-O2. These results suggest that the 5,7-dihydroxyflavone in combination with TRAIL might be used for cancer prevention and/or therapy.

The molecular mechanism of luteolin-induced apoptosis is potentially related to inhibition of angiogenesis in human pancreatic carcinoma cells.

Luteolin has been shown to have a strong anticancer effect on various cancer models via programmed cell death (apoptosis). However, the fundamental mechanisms of these effects are still unclear. In the present study, we examined the question of whether or not luteolin can inhibit proliferation of pancreatic carcinoma cells, via apoptosis. We used three human pancreatic carcinoma cell lines, PANC-1, CoLo­357 and BxPC-3 in our study. In luteolin-treated pancreatic carcinoma cells, typical features of apoptosis were observed. Luteolin increased the expression of the pro-apoptotic protein Bax and decreased the expression of the anti-apoptotic protein Bcl-2, with a concomitant increase in the levels of caspase-3 and cleaved PARP after treatment for 24 h. Luteolin inhibited HUVEC proliferation and vessel growth in CAM in vivo. In addition, the concentration of VEGF in the conditioned medium from human pancreatic carcinoma cells was downregulated by luteolin. Pancreatic carcinoma cells, pretreated with luteolin, could decrease the capillary-like structure formation by HUVEC, which was analyzed by a co-culture system. The abatement of VEGF secretion was related to the inhibition of VEGF mRNA expression, which may be regulated by inhibiting the transcription activity of nuclear transcription factor NF-κB.

Analgesic-antitumor peptide inhibits proliferation and migration of SHG-44 human malignant glioma cells.

Malignant gliomas, the most common subtype of primary brain tumors, are characterized by high proliferation, great invasion, and neurological destruction and considered to be the deadliest of human cancers. Analgesic-antitumor peptide (AGAP), one of scorpion toxic polypeptides, has been shown to have antitumor activity. Here, we show that recombinant AGAP (rAGAP) not only inhibits the proliferation of gliomas cell SHG-44 and rat glioma cell C6, but also suppresses the migration of SHG-44 cells during wound healing. To explain these phenomena, we find that rAGAP leads to cell cycle of SHG-44 arrested in G1 phase accompanied by suppressing G1 cell cycle regulatory proteins CDK2, CDK6, and p-RB by means of the down-regulated protein expression of p-AKT. Meanwhile, rAGAP significantly decreases the production of NF-κB, BCL-2, p-p38, p-c-Jun, and p-Erk1/2 and further suppresses the activation of VEGF and MMP-9 in SHG-44 cells. These findings suggest rAGAP inhibit proliferation and migration of SHG-44 cells by arresting cell cycle and interfering p-AKT, NF-κB, BCL-2, and MAPK signaling pathways.

Luteolin induced G2 phase cell cycle arrest and apoptosis on non-small cell lung cancer cells.

In this study, we investigated the underlying molecular mechanism for the potent cell cycle inhibition and pro-apoptotic effect of luteolin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-chromenone) on human non-small-cell lung carcinoma cell line A549. MTT assay showed that luteolin had obvious cytotoxicity on A549 with IC(50) of 40.2 µM at 48 h. Pro-apoptotic effect of luteolin on A549 cells was demonstrated by Hoechst 33258 staining assay and annexin V-FITC/PI double staining analysis. A great quantity of apoptotic cells and increasing G2 phase cells were observed by flow cytometry. Western blotting assay revealed that luteolin activated JNK, increased Bax, promoted procaspase-9 cleavage and activated caspase-3 at last. Assay using TNFα, an active agent of NF-κB, showed that pretreatment of A549 cells with luteolin could inhibit TNFα induced trans-nuclear of NF-κB. In summary, luteolin displayed a significant cytotoxic effect through cell cycle arrest and apoptosis induction in A549 cells. Pro-apoptotic effect was implemented via activating JNK and inhibiting translocation of NF-κB (p65). These results suggested that luteolin might have therapeutic potential against NSCLC.

Expression and purification of an antitumor-analgesic peptide from the venom of Mesobuthus martensii Karsch by small ubiquitin-related modifier fusion in Escherichia coli.

To prevent protein aggregation, some proteins are usually expressed as fusion proteins from which target proteins can be released by proteolytic or chemical reagents. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the antitumor-analgesic peptide from the venom of Buthus martensii (Karsch) scorpion (AGAP). The optimal expression level of the soluble fusion protein, SUMO-AGAP, was up to 40% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease (Ulp1) to obtain the recombinant AGAP (rAGAP), which was further purified by Ni-NTA affinity chromatography. The purified final product was >95% pure by SDS-PAGE stained with Coomassie brilliant blue R-250. Mass spectroscopic analysis indicated the protein to be 7142.63 Dalton, which equaled the theoretically expected mass. N-terminal sequencing of rAGAP showed the sequence corresponded to the native protein. MTT assay indicated the rAGAP could significantly inhibit the proliferation of Jurkat and Hut 78 T lymphoma cell lines. The further writhing experiment showed that the rAGAP had an intensive analgesic effect. The expression strategy presented in this study allows convenient high yield and easy purification of the rAGAP with native sequences.

[Auricularia auricular polysaccharide protects myocardium against ischemia/reperfusion injury].

OBJECTIVE: To determine whether auricularia auricular polysaccharide (AAP) protects heart against ischemia/reperfusion (1/ R) injury and its underlying mechanisms. METHODS: Male Sprague-Dawley rats, pretreated with AAP (50, 100, 200 mg/(kg x d), gastric perfusion) for 4 weeks, were used for Langendorff isolated heart perfusion. The hearts were subjected to global ischemia for 30 min followed by 120 min of reperfusion and the left ventricular hemodynamic parameters were measured. Formazan, a product of 2, 3, 5-triphenyl-tetrazolium chloride (TTC), which is proportional to myocardial viability, was measured at 490 nm, and the level of lactate dehydrogenase (LDH) in the coronary effluent was measured to evaluate the cardiac injury. The cardiac malondialdehyde (MDA), a product of lipid peroxidation, and superoxide dismutase (SOD) activity were determined after myocardial I/R. RESULTS: The pretreatment with AAP at 50, 100, 200/(kg d) for 4 weeks before I/R increased myocardial formazan content, reduced LDH release, improved the recovery of the left ventficular developed pressure, maximal rise rate of left ventricular pressure, and rate pressure product (left ventricular developed pressure multiplied by heart rate) attenuated the decrease of coronary flow during reperfusion. The cardiac protective effect of high dose AAP was more potent than that of compound radix salviae miltiorrhizae (CRSM, 4 ml/(kg x d), gastric perfusion for 4 weeks). Pretreatment with AAP (100 mg/(kg x d)) markedly inhibited the increase of MDA level and the decrease of SOD activity induced by I/R in myocardium. CONCLUSION: The findings indicate that in the isolated rat heart, AAP protects myocardium against ischemia/reperfusion injury via enhancing the activity of SOD and reducing lipid peroxidation in heart.

[Study of the effects of Auricularia auricular polysaccharide on local ischemia/reperfusion injury in rat].

OBJECTIVE: To investigate the influence of Auricularia Auricular polysaccharide (APP) on acute cerebral injury induced by ischemia/reperfusion in rats and its underlying mechanism. METHODS: Adult male SD rats were intragastrically pretreated with AAP at a low (50 mg/kg) or high (100 mg/kg) dose once a day for 20 days before operation. Rats intraperitoneally injected with ginkgo biloba extract (EGb671) were taken as positive control. Focal ischemia was achieved by middle cerebral artery occlusion (MCAO) on the right side for 60 min. After 24 hrs of reperfusion, the nerve function defects were recorded by Longa's score and the brain infarct sizes were measured by 2,3,5-Triphenyl-tetrazolium-chlor (TTC) staining. Apoptotic neurons were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining after 48 h of reperfusion. The levels of oxidative stress was determined via the mitochondria-generated reactive oxygen species (ROS). RESULTS: AAP treatment decreased Longa's score, brain infarct size, apoptotic neurons and mitochondria-generated ROS in a dose-dependent manner. AAP at 100 mg/kg gave a better performance compared with EGb671 on all parameters examined. CONCLUSION: AAP treatment protected rat brain from focal ischemia/reperfusion injury by its anti-oxidative effect and worked better than EGb671.

High-level expression and characterization of an anti-VEGF165 single-chain variable fragment (scFv) by small ubiquitin-related modifier fusion in Escherichia coli.

Antibodies currently constitute the most rapidly growing class of human therapeutics; however, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. High expression of active single-chain antibody fragment (scFv) in Escherichia coli has not been successful, as the protein contains three intramolecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the scFv gene against VEGF165 with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO-scFv fusion gene that was highly expressed in the BL21(DE3) strain. The optimal expression level of the soluble fusion protein, SUMO-scFv, was up to 28.5% of the total cellular protein. The fusion protein was purified by Ni nitrilotriacetic acid (NTA) affinity chromatography and cleaved by a SUMO-specific protease to obtain the native scFv, which was further purified by Ni-NTA affinity chromatography. The result of the high-performance liquid chromatography showed that the purity of the recombinant cleaved scFv was greater than 98%. The primary structure of the purified scFv was confirmed by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analysis. In vitro activity assay demonstrated that the recombinant scFv could dose-dependently inhibit VEGF165-induced human umbilical vein-derived endothelial cell proliferation. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant scFv with native sequences.

WITHDRAWN: High-level expression of recombinant human vascular endothelial growth factor (rhVEGF165) in silkworm larvae (Bombyx mori).

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Expression, purification, and characterization of recombinant human soluble BAFF secreted from the yeast Pichia pastoris.

The B lymphocyte stimulator (BAFF) is a novel member of the tumor necrosis factor (TNF) ligand family which is important in B lymphocyte maturation and survival. Herein, the cDNA coding for the extracellular domain of the BAFF (hsBAFF) has been cloned into the secreting expression organism Pichia pastoris. SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hsBAFF, a 20.2 kDa glycosylated protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using DEAE-Sepharose ion exchange and Superdex 75 size-exclusion chromatography steps. Finally, 102 mg of the protein was obtained in high purity from 1 L of the supernatant and its identity to hsBAFF was confirmed by NH(2)-terminal amino acid sequence analysis Bioactivity of the recombinant hsBAFF was confirmed by the ability of the protein to stimulate human B lymphocyte proliferation in vitro. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional hsBAFF for both research and industrial purpose.

[Cardioprotective effect of ischemic postconditioning and preconditioning against prolonged ischemia and reperfusion induced injury in isolated rat heart].

OBJECTIVE: To explore the cardioprotection effect of co-treatment with ischemic postconditioning and preconditioning in ischemia/reperfusion (I/R) injury and the related mechanism. METHODS: Male Sprague-Dawley rats were used for Langendorff isolated heart perfusion. The hearts were subjected to global ischemia for 60 min followed by 120 min of reperfusion. The cardiomyocyte viability was measured by MTT-formazan method, and the cardiac injury was evaluated by the levels of lactate dehydrogenase (LDH) in the coronary effluent. Ventricular hemodynamic parameters were also measured. RESULT: In 60 min of ischemia and 120 min of reperfusion group, ischemic postconditioning increased formazan content, reduced LDH release, but hemodynamic parameters did not improved. Co-treatment with ischemic postconditioning and preconditioning during the prolonged ischemia further improved the hemodynamic parameters. The calcium activated potassium channel antagonist paxilline attenuated the effect of co-treatment with ischemic postconditioning and preconditioning. CONCLUSION: Ischemic postconditioning and preconditioning may synergically protect myocardium from severe ischemia injury, which may be related to calcium-activated potassium channel.

[Kappa-opioid receptor mediates the cardioprotective effect of ischemic postconditioning].

OBJECTIVE: To investigate the effect of kappa-Opioid receptors in the cardioprotection elicited by ischemic postconditioning and the underlying mechanism. METHODS: The isolated perfused hearts of male Sprague-Dawley rats were subjected to 30 min of global ischemia followed by 120 min of reperfusion. formazan content of myocardium was measured spectrophotometrically, and the level of lactate dehydrogenase (LDH) in the coronary effluent was also measured. In isolated ventricular myocytes hypoxia postconditioning was achieved by 3 cycles of 5 min reoxygenation/5 min hypoxia starting at the beginning of reoxygenation, and cell viability was measured. RESULT: In the Langendorff perfused rat heart model, ischemic postconditioning (6 cycles of 10 s reperfusion/10 s global ischemia starting at the beginning of reperfusion) increased formazan content, reduced LDH release, improved the recovery of the left ventricular developed pressure, maximal rise/fall rate of left ventricular pressure, left ventricular end-diastolic pressure and rate pressure product (left ventricular developed pressure multiplied by heart rate), attenuated the decrease of coronary flow during reperfusion and increased the isolated cell viability. Pretreatment with nor-BNI, an antagonist of kappa-Opioid receptors and mitoK(ATP) blocker 5-HD attenuated the effect of ischemic/hypoxic postconditioning. CONCLUSION: Postconditioning may protect myocardium against ischemia/reperfusion injury via activating kappa-Opioid receptors and mitoK(KATP).

[The alterations of nitric oxide synthase activity of ventricular cardiac muscle of rats in two septic shock models].

AIM: To observe the differences of hemodynamics and nitric oxide synthase(NOS) activity of ventricular cardiac muscle in two septic shock models and explore the possible mechanism. METHODS: Two rat models of septic shock[lipopolysaccharide(LPS)-induced and cecal ligation and puncture (CLP)-induced septic shock] were used. The hemodynamic parameters and nitric oxide synthase activity of ventricular cardiac muscle were measured. RESULTS: The hemodynamic parameters in CLP-induced model were increased in the early stage and decreased in the late stage while in LPS-induced model the parameters showed the same change of the CLP late stage. Both LPS model and CLP model (late stage) showed significant increase in NOS activity, but there was no difference between the two models. After treatment of the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME), the parameters of CLP-late stage and LPS model increased significantly. The NOS activity reached the highest level in the CLP-middle stage. The production of nitrite/nitrate decreased significantly in LPS model and CLP model(late stage) after treatment of L-NAME, but the nitrite/nitrate produced by constitutive NOS in LPS model was higher than CLP model(late stage). CONCLUSION: The increase of the NOS activity may be the main reason to lead to the depression of the hemodynamic parameters. Inducible NOS may play the leading role in the LPS model while cNOS and iNOS have the same effect in the CLP model.

[Experimental studies on antiarrhythmic effect of jumi extraction].

AIM: To investigate the antiarrhythmic effect of jumi (JM) extraction. METHODS: The conventional antiarrhythmic methods were used. RESULTS: Administration of JM extraction reduced the occurrence of ventricular fibrillation induced by chloroform in a dose-dependent manner in mice. Quinidine significantly decreased the number of ventricular premature beats and ventricular tachycardia, shortened the duration of arrhythmia in aconitine-treated rats. But JM extraction had no effect on aconitine-induced arrhythmia. Compared with control, arrhythmia score was lower in ischemia/reperfusion rats which pretreated with 2.0 g/kg of JM extraction. CONCLUSION: JM extraction has obvious protection effects in chloroform- and ischemia-induced arrhythmia, but has no effect in aconitine-induced arrhythmia.

[Role of mitochondrial calcium uniporter in myocardial hypoxia/reoxygenation induced injury].

AIM: To investigate the role and mechanism of mitochondrial calcium uniporter (MCU) in myocardial hypoxia/reoxygenation injury. METHODS: Isolated rat hearts were perfused with Langendorff apparatus. The hypoxia/reoxygenation injury was achieved by ligation of left anterior coronary artery for 30 min followed by release of ligation for 120 min. The left ventricular developed pressure (LVDP), the maximum rise/fall rate of left ventricular pressure (+/- dP/dt(max)), and the left ventricular end-diastolic pressure (LVEDP) were recorded. Activities of lactate dehydrogenase (LDH) in coronary effluent and reactive oxygen species (ROS) of myocardial mitochondria were spectrophotometrically assayed. Infarct size was determined by TTC staining method. RESULTS: Compared with the hypoxia/reoxygenation (H/R) group, ruthenium red (RR, 5 micromol/L), given at the on set of reoxygenation, significantly improved the contractile function of left ventricle, decreased the myocardial infarct size, alleviated the production of ROS in myocardial mitochondria and LDH release in coronary effluent. Spermine (20 micromol/L), given at the onset of reoxygenation, enhanced the production of ROS in the mitochondria and LDH release in coronary effluent at 5, 20 and 30 min of reoxygenation, however, there were no significant differences of ventricular contractile parameters and infarct size between groups subjected to hypoxia/reoxygenation with or without spermine treatment. Co-treatment of ROS scavenger N-2-mercaptopropionyl glycine (1 mmol/L) with spermine abolished the effect of spermine. CONCLUSION: Inhibition of mitochondrial calcium uniporter may refrain heart from hypoxia/reoxygenation injury via decreasing the production of ROS in heart mitochondria.

[Cardioprotection of mitoSlo1 channel activation involves mitochondrial permeability transition in ischemia and reperfusion of rat hearts].

OBJECTIVE: To investigate whether the cardioprotection of mitochondrial Slo channel (mitoSlo(1) channel) is associated with mitochondrial permeability transition in isolated rat hearts subjected to ischemia and reperfusion. METHODS: Isolated perfused rat hearts were subjected to 30 min regional ischemia (occlusion of left anterior descending artery) and 120 min reperfusion. The infarct size, lactate dehydrogenase (LDH) release during reperfusion and ventricular hemodynamic parameters were measured. RESULTS: Pretreatment with mitoSlo(1) channel opener, NS1619 10 micromol/L for 10 min reduced the infarct size and LDH release, and improved the recovery of left ventricular developed pressure, left ventricular end-diastolic pressure, maximal rise/fall rate of left ventricular pressure and coronary flow during reperfusion. Administration of atractyloside (20 micromol/L), an opener of mitochondrial permeability transition pore, for 20 min (last 5 min of ischemia and first 15 min of reperfusion) attenuated the reduction of infarct size and LDH release and improvement of left ventricular performance induced by NS1619. In the isolated mitochondria, a significant inhibition of Ca(2+)-induced swelling was observed when mitochondria were incubated with NS1619. CONCLUSION: MitoSlo(1) channel activation by NS1619 protects the myocardium against ischemia and reperfusion injury by inhibiting mitochondrial permeability transition pore opening.