Analgesic activity of myricetin isolated from Myrica rubra Sieb. et Zucc. leaves.

Myrica rubra Sieb. et Zucc. leaves are commonly used as an astringent, antidiarrheic, and analgesics in folk medicine in China. In the present study, the analgesic activity of myricetin, a major compound in Myrica rubra Sieb. et Zucc. leaves was evaluated in vivo. The analgesic effect of myricetin was tested by a serial of models, such as acetic acid-induced writhing response, formalin-induced paw licking and hot plate test. The sedative activity was evaluated by pentobarbital-induced sleep time. Platelet aggregation induced by collagen and arachidonic acid was also performed in vitro. Myricetin showed a significant inhibition on chemical nociceptive models such as the acetic acid-induced writhing response and the licking time on the late phase in the formalin test in a dose-dependent manner, but did not manifest a signicant effect in hot plate test. Myricetin was also not able to increase the sleeping time induced by pentobarbital, which further indicated that the analgesic effect of myricetin was unrelated to sedation. In addition, myricetin inhibited the content of PGE2 in the peritoneal fluid and platelet aggregation induced by collagen and arachidonic acid in vitro. These results collectively demonstrated that myricetin possessed potent analgesic activity, which was related with peripheral analgesia, but, not with the opioid system. Myricetin may be a potent COX-1 inhibitor with anti-platelet activity.

Chronic high-dose morphine treatment promotes SH-SY5Y cell apoptosis via c-Jun N-terminal kinase-mediated activation of mitochondria-dependent pathway.

Chronic high doses of morphine inhibit the growth of various human cancer cell lines. However, the mechanisms by which such high-dose morphine inhibits cell proliferation and induces cell death are not fully understood. Here we show that c-Jun N-terminal kinase (JNK) plays a pivotal role in high-dose morphine-induced apoptosis of SH-SY5Y cells in a mitochondria-dependent manner. Activation of JNK by morphine led to reactive oxygen species (ROS) generation via the mitochondrial permeability transition pore, because the mPTP inhibitor cyclosporin A significantly inhibited ROS generation. ROS in turn exerted feedback regulation on JNK activation, as shown by the observations that cyclosporin A and the antioxidant N-acetylcysteine significantly inhibited the phosphorylation of JNK induced by morphine. ROS-amplified JNK induced cytochrome c release and caspase-9/3 activation through enhancement of expression of the proapoptotic protein Bim and reduction of expression of the antiapoptotic protein Bcl-2. All of these effects of morphine could be suppressed by the JNK inhibitor SP600125 and N-acetylcysteine. The key role of the JNK pathway in morphine-induced apoptosis was further confirmed by the observation that decreased levels of JNK in cells transfected with specific small interfering RNA resulted in resistance to the proapoptotic effect of morphine. Thus, the present study clearly shows that morphine-induced apoptosis in SH-SY5Y cells involves JNK-dependent activation of the mitochondrial death pathway, and that ROS signaling exerts positive feedback regulation of JNK activity.

Pharmacological characterization of ATPM [(-)-3-aminothiazolo[5,4-b]-N-cyclopropylmethylmorphinan hydrochloride], a novel mixed kappa-agonist and mu-agonist/-antagonist that attenuates morphine antinociceptive tolerance and heroin self-administration behavior.

ATPM [(-)-3-amino-thiazolo[5,4-b]-N-cyclopropylmethylmorphinan hydrochloride] was found to have mixed kappa- and mu-opioid activity and identified to act as a full kappa-agonist and a partial mu-agonist by in vitro binding assays. The present study was undertaken to characterize its in vivo effects on morphine antinociceptive tolerance in mice and heroin self-administration in rats. ATPM was demonstrated to yield more potent antinociceptive effects than (-)U50,488H (trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide). It was further found that the antinociceptive effects of ATPM were mediated by kappa- and mu-, but not delta-opioid, receptors. In addition to its agonist profile on the mu-receptor, ATPM also acted as a mu-antagonist, as measured by its inhibition of morphine-induced antinociception. It is more important that ATPM had a greater ratio of the ED(50) value of sedation to that of antinociception than (-)U50,488 (11.8 versus 3.7), indicative of a less sedative effect than (-)U50,488H. In addition, ATPM showed less potential to develop antinociceptive tolerance relative to (-)U50,488H and morphine. Moreover, it dose-dependently inhibited morphine-induced antinociceptive tolerance. Furthermore, it was found that chronic treatment of rats for 8 consecutive days with ATPM (0.5 mg/kg s.c.) produced sustained decreases in heroin self-administration. (-)U50,488H (2 mg/kg s.c.) also produced similar inhibitory effect. Taken together, our findings demonstrated that ATPM, a novel mixed kappa-agonist and mu-agonist/-antagonist, could inhibit morphine-induced antinociceptive tolerance, with less potential to develop tolerance and reduce heroin self-administration with less sedative effect. kappa-Agonists with some mu-activity appear to offer some advantages over selective kappa-agonists for the treatment of heroin abuse.

[Rapid pharmacokinetics screening of drug candidates in vitro and in vivo].

The paper is to report the pharmacokinetic character of a series of chemical compounds in vitro and in vivo. Metabolism stability of a series of chemical compounds was screened by using rat liver microsomes. The samples of different chemical compounds were combined and then simultaneously detected by LC-MS/MS. Compounds y13, y12 and y11 were screened out by microstability assay in vitro. The pharmacokinetics of compounds y11, y12 and y13 was evaluated by using SD rat. The plasma samples were pooled at the same time. The plasma concentrations were determined by LC-MS/MS. The pharmacokinetic character of two compounds y13, y11 was good by screening in vivo, so they were developed for further research. High-throughput screening of drug candidates in vitro and in vivo was effective, to provide information for the chemical structure information and lower the drug development risk.

Neuroprotection of ginsenoside Re in cerebral ischemia-reperfusion injury in rats.

In the present study, we have investigated the neuroprotective potential of ginsenoside Re (Re) in the middle cerebral artery occlusion model in Sprague-Dawley rats. Adult male Sprague-Dawley rats were treated with Re (5, 10 or 20 mg kg(- 1), P.O. for 7 days, once a day) prior to occlusion. There was a significant increase in the neurological symptoms in ischemic animals as compared with the sham group animals. These effects were attenuated by 10 and 20 mg kg(- 1) Re, P.O. There was a significant increase in the level of malondialdehyde (MDA) in ischemic animals indicating oxidative stress. An elevated level of MDA in ischemic animals was reduced by 10 and 20 mg kg(- 1) Re, P.O., respectively. It was observed that Re significantly decreased mitochondrial swelling, thereby preventing the reduction of H(+)-ATPase activity. This study demonstrates the neuroprotective potential of Re in cerebral ischemia-reperfusion injury in rats.

Combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and canstatin gene suppression therapy on breast tumor xenograft growth in mice.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy and canstatin gene therapy have been investigated extensively in human xenograft tumor models established in immunocompromised nude mice. However, combination antitumor activity of these two agents and the safety of such gene constructs driven by the human telomerase reverse transcriptase (hTERT) promoter in nude mice have not been well documented. We hypothesized that TRAIL and canstatin gene therapy driven by the hTERT promoter might overcome the problem of liver toxicity and still effectively induce apoptosis on tumor cells. In this study, we evaluated the antitumor effects of TRAIL in human breast cancer cell lines and the antiangiogenic effects of canstatin on ECV204 cells. We also analyzed the effects of combined gene therapy using both TRAIL and canstatin in a human breast cancer nude mouse model. Tumor growth, tumor inhibition rate of each group, and toxicity were evaluated after gene therapy. Our results demonstrate that treatment using the canstatin- or TRAIL-expressing vector alone significantly suppresses tumor growth, compared to PBS or a vector control. We also found that combining these two therapies had greater antitumor activity than either treatment alone in the mouse model. Moreover, induction of apoptosis was not detected in normal mouse tissues after intratumoral injection of vectors and liver toxicity did not occur with either treatment. Thus, the combination of TRAIL and canstatin appears to be a promising approach for the gene therapy of breast tumors.

The survival time post-cecal ligation and puncture in sinoaortic denervated rats.

Arterial baroreflex (ABR) function is an important determinant factor in prognosis of many cardiovascular diseases. The present work was designed to study the relationship between ABR function and the survival time of septic shock in a cecal ligation and puncture (CLP) rat model. The dysfunction of ABR was introduced by sinoaortic denervation (SAD). It was found that the survival time after CLP was significantly reduced in SAD rats compared with sham-operated rats (12.7 +/- 2.92 hours versus 15.0 +/- 4.01 hours; P < 0.05). Furthermore, significant differences were also seen when the results were expressed by Kaplan-Meier survival curves. Compared with the baseline values, both noradrenaline and adrenaline significantly increased in both SAD and Sham groups after CLP, but we found the baseline of noradrenaline was significantly elevated in SAD rats. In addition, the TNF-alpha, noradrenaline, and adrenaline levels of the SAD group were significantly higher than those of the Sham group at 5 hours post-CLP. In conclusion, the present work demonstrates that ABR function was related to the survival time in CLP-induced lethal shock model. The loss of inhibition in the sympathetic activity and in the release of some inflammatory cytokines during CLP-induced septic shock related to baroreflex and/or chemoreflex dysfunction may be the mechanisms involved in the poorer prognosis in septic shock.

Apolipoprotein C-II promoter T-->A substitution at position -190 affects on the transcription of the gene and its relationship to hyperlipemia.

A Chinese patient with severe hypertriglyceridemia was found to have similar clinical features to that of malignant hyperlipemia in infancy. DNA sequence analysis of the apoC-II gene from the patient's parents revealed a novel heterozygous mutation of T-->A substitution at position -190 base in the apoC-II promoter. We speculated that the patient was a homozygote of the same mutation that resulted in the deficiency of apoC-II. In vitro expression studies showed T-->A substitution in the apoC-II promoter leads to a decrease by approximately 20% in transcriptional activity compared with its counterpart that inserted the normal promoter. These results suggested that T-->A substitution at position -190 in the apoC-II gene promoter only partly affected transcriptional activity of the apoC-II promoter, leading to decrease of apoC-II expression in quantity.

Blockade of TRAIL pathway ameliorates HBV-induced hepatocyte apoptosis in an acute hepatitis model.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) may play important roles during hepatitis B virus (HBV) infection. In this study, we used a recombinant human soluble death receptor 5 (sDR5) to explore its effect in a mouse model of HBV-induced acute hepatitis. By measuring blood transaminase activity and hepatocyte apoptosis, we found that sDR5 could alleviate liver damage by blocking TRAIL-induced apoptosis of HBV-transfected hepatocytes. sDR5 injection at 16 mg/kg 24h before HBV transfection was the most effective. Additionally, we showed that sDR5 was equally effective in protecting liver injury as the Stronger Neo-Minophagen C (SNMC), a commonly used drug for patients with liver diseases. Thus, sDR5 represents a potential novel therapeutic drug for patients with fulminant hepatitis.

Protective effect of ginsenoside-Re against cerebral ischemia/reperfusion damage in rats.

To investigate the protective effect of ginsenoside Re (Re) against cerebral ischemia-reperfusion injury, adult male Wistar rats weighing 250-300 g were subjected to either sham surgery or middle cerebral artery occlusion (MCAO) for 2 h of brain ischemia and 2 h reperfusion. A fluorescence polarization assay was carried out for membrane fluidity of brain mitochondria. Lipid peroxidation [malondiadehyde (MDA) formation], superoxide dismutase (SOD) and glutathion peroxidase (GSH-Px) of rat brain were estimated by fluorometric methods. It was observed that Re (5, 10, 20 mg kg-1 p.o. pretreatment for 7 d, once a day) significantly improved the fluidity of mitochondrial membranes as demonstrated by a reduction of average microviscosity, ameliorated lipid peroxidation by raising the activities of SOD and GSH-Px, and reduced the content of MDA in rat brain. This study demonstrated a direct protective effect of Re against cerebral ischemia-reperfusion injury.

A novel dual peroxisome proliferator-activated receptors alpha and gamma agonist with beneficial effects on insulin resistance and lipid metabolism.

Peroxisome proliferator-activated receptors (PPARs) alpha and gamma are key regulators of lipid homeostasis and insulin resistance. In this study we show that a novel compound, 3-{4-[2-(5-methyl-2-phenyl-oxazol-4-yl)-ethoxy]-phenyl}- 2-[2-(2-nitro-phenoxy)-acetyl amino]-propionic acid (O325H), is an agonist with dual effect on PPARalpha/gamma by using dual-luciferase reporter gene assay. By activating PPARalpha and PPARgamma simultaneously, O325H promotes pre-adipocyte differentiation and up-regulates the expression of glucose and lipid metabolic target genes. In diabetic mice, administration of O325H at 10 mg/kg decreases the blood lipid and glucose levels. Therefore, O325H has dual action on PPARalpha and PPARgamma and is a promising agent for the amelioration of lipid metabolic disorders and diabetes associated with insulin resistance.

C333H, a novel PPARalpha/gamma dual agonist, has beneficial effects on insulin resistance and lipid metabolism.

AIM: To examine the effects of novel peroxisome proliferator-activated receptor (PPAR) alpha/gamma dual agonist C333H on insulin resistance and lipid metabolism. METHODS: An established dual-luciferase reporter gene assay system was used in vitro to test the activity of C333H with respect to the transcription of human PPARalpha and PPARgamma. A preadipocyte differentiation assay and reverse transcription-polymerase chain reaction were used to detect the functional activities of C333H. In db/db mice, the effects of C333H were investigated with respect to lowering of blood glucose and lipid levels. RESULTS: C333H was determined to be a novel PPARalpha/gamma dual agonist because it strongly induced luciferase activity on human PPARalpha and PPARgamma, promoting the differentiation of preadipocytes to adipocytes, and functioning in upregulating the expression of some glucose and lipid metabolic target genes of the PPAR. In addition, C333H efficiently reduced blood lipid and glucose concentrations in db/db diabetic mice. CONCLUSION: C333H has dual action on both PPARalpha and PPARgamma, and might be of interest for the amelioration of lipid metabolic disorders and insulin resistance associated with type 2 diabetes.

C16, a novel advanced glycation endproduct breaker, restores cardiovascular dysfunction in experimental diabetic rats.

AIM: Advanced glycation endproducts (AGE) have been implicated in the pathogenesis of diabetic complications, including diabetic cardiovascular dysfunction. 3-[2-(4-Bromo-phenyl)-1-methyl-2-oxo-ethyl]-4,5,6,7-tetrahydro-benzothiazol-3-ium bromide (C16), a novel AGE breaker, was investigated for its effects on the development of cardiovascular disease in diabetic rats. METHODS: Rats that had streptozotocin-induced diabetes for 12 weeks were divided into groups receiving C16 or vehicle by gavage. RESULTS: In hemodynamic studies of the left ventricle, C16 treatment (25 or 50 mg/kg) for 4 weeks resulted in a significant increase in left ventricular systolic pressure, +dp/dt(max), and -dp/dt(max) as compared with vehicle-treated diabetic rats. Furthermore, in hemodynamic studies of the cardiovascular system, C16 (12.5, 25, or 50 mg/kg) treatment for 4 weeks resulted in a dose-dependent and significant increase in cardiac output, a reduction of total peripheral resistance, and an increase in systemic arterial compliance when compared with vehicle-treated diabetic rats. Biochemical studies showed that C16 treatment also resulted in a significant decrease in immunoglobulin G-red blood cell surface crosslink content and an increase in collagen solubility. Morphological and immunohistochemical examinations indicated that C16 was able to prevent increases of the collagen type III/I ratio in the aorta and decrease the accumulation of AGE in the aorta. CONCLUSION: C16 has the ability to reduce AGE accumulation in tissues in vivo, and can restore diabetes-associated cardiovascular disorders in rats. This provides a potential therapeutic approach for cardiovascular disease associated with diabetes and aging in humans.

Possible mechanisms of the protection of ginsenoside Re against MPTP-induced apoptosis in substantia nigra neurons of Parkinson's disease mouse model.

We have investigated the role of ginsenoside Re (Re) in preventing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced apoptosis of the substantia nigra neurons in the mouse model of Parkinson's disease (PD). C57BL mice have been administrated i.s.c. with MPTP to establish the PD model. Pretreatment groups were given different doses of Re (6.5, 13, 26 mg kg(-1)) i.g. for 13 days. Transmission electron microscope (TEM), tyrosine hydroxythase (TH) immunostaining and TDT-mediated dUTP nick-end labeling (TUNEL) staining have been used to observe the damage of substantia nigral neurons. To measure the expression of inducible nitric oxide synthase (iNOS), Bcl-2, Bax protein and expression of Bcl-2, Bax gene, immunohistochemistry and in situ hybridization have been explored respectively. Western blot analysis has been performed with anti-caspase-3. Pretreatment with Re (13, 26 mg kg(-1)) markedly increases TH-positive neurons and decreases the TUNEL-positive ratio compared with the MPTP model group. Furthermore, Re could enhance the expression of Bcl-2 protein and Bcl-2 mRNA, but reduce the expression of Bax, Bax mRNA, and iNOS, and weaken the cleavage of caspase-3. In summary, ginsenoside Re showed protection from MPTP-induced apoptosis in the PD model mouse nigral neurons and this effect may be attributable to upregulating the expression of Bcl-2 protein, downregulating the expression of Bax, and iNOS protein, and inhibiting the activation of caspase-3.

Human endostatin gene transfer, either naked or with liposome, has the same inhibitory effect on growth of mouse liver tumor cells in vivo.

AIM: To explore a safe and efficient strategy of tumor therapy using anti-angiogenetic agents. METHODS: Endostatin gene with a signal sequence of human IgGgamma chain was amplified by PCR and cloned into pVAX1 plasmid which was the only vector authorized by FDA in clinical trial to construct a recombinant plasmid named as pVAX-sEN. The recombinant plasmid was detected with EcoRI/KpnI and DNA sequencing. BALB/c mice bearing hepatocarcinoma cell line H22 were treated with naked pVAX-sEN or liposome-DNA complex in which the dose of DNA and the ratio of DNA and liposome were different from each other. To compare the efficiency of gene transfection, expression of endostatin at the treated tumor site was assayed with ELISA. To investigate the effect of pVAX1-sEN on hepatocellular carcinoma, pVAX-sEN either naked or in liposome-DNA complex was injected into BALB/c mice bearing H22, then the diameter of tumors was measured, microvessel density was detected by immunohistochemistry, endostatin expression in vivo was assayed at different time points. RESULTS: DNA sequencing showed the endostatin gene with the signal peptide was correctly cloned. In situ gene expression assay indicated that both the ratio of DNA and liposome and the dose of DNA could affect the gene transfection efficiency. Interestingly, naked pVAX-sEN had a similar in situ endostatin expression to pVAX-sEN with liposome. Animal experiments showed that pVAX-sEN together with pVAX-sEN-liposome complex could efficiently suppress the growth of mouse hepatoma cells. CONCLUSION: Naked endostatin plasmid intratumoral injection can get a similar gene transfection efficiency to liposome-DNA complex when used in situ.

Effect of (3,5,6-trimethylpyrazin-2-yl)methyl 2-[4-(2-methylpropyl)phenyl]propanoate (ITE), a newly developed anti-inflammatory drug, on type II collagen-induced arthritis in mice.

The effect of (3,5,6-trimethylpyrazin-2-yl)methyl 2-[4-(2-methylpropyl)phenyl]propanoate (ITE) on type II collagen (CII)-induced arthritis in mice was studied. Mice were immunized twice with CII, ITE being given orally once a day for 40 d after the 1st immunization. Clinical assessment showed that ITE had no effect on the day of onset of arthritis but did lowered the incidence rate of arthritis and the arthritis score. And ITE had a marked suppressive effect on the mouse hind paw edema induced by CII. ITE suppressed the delayed-type mouse ear skin reaction to CII but had no effect on the level of serum anti-CII antibodies. These results suggest that ITE inhibits the development of CII-induced arthritis in mice by suppressing delayed-type hypersensitivity to CII.

Establishment of mice model with human viral hepatitis B.

AIM: To establish a mice model of hepatitis B by using HBV-transgenic mice, and to transfer HBV-specific cytotoxic T lymphocytes (CTL) induced from syngeneic BALB/c mice immunized by a eukaryotic expression vector containing HBV complete genome DNA. METHODS: HBV DNA was obtained from digested pBR322-2HBV and ligated with the vector pcDNA3. Recombinant pcDNA3-HBV was identified by restriction endonuclease assay and transfected into human hepatoma cell line HepG2 with lipofectin. ELISA was used to detect the expression of HBsAg in culture supernatant, and RT-PCR to determine the existence of HBV PreS1 mRNA. BALB/c mice were immunized with pcDNA3-HBV or pcDNA3 by intramuscular injection. ELISA was used to detect the expression of HBsAb in serum. MTT assay was used to measure non-specific or specific proliferation ability and specific killing activity of spleen lymphocytes. Lymphocytes from immunized mice were transferred into HBV-transgenic mice (2.5X10(7) per mouse). Forty-eight hours later, the level of serum protein and transaminase was detected with biochemical method, liver and kidney were sectioned and stained by HE to observe the pathological changes. RESULTS: By enzyme digestion with Eco RI, Xho I and Hind III, the recombinant pcDNA3-HBV was verified to contain a single copy of HBV genome, which was inserted in the positive direction. HepG2 cells transfected with the recombinant could stably express PreS1 mRNA and HBsAg. After immunized by pcDNA3-HBV for 4 weeks, HBsAb was detected in the serum of BALB/c mice. The potential of spleen lymphocytes for both non-specific and specific proliferation and the specific killing activity against target cells were enhanced. The transgenic mice in model group had no significant changes in the level of serum protein but had an obvious increase of ALT and AST. The liver had obvious pathological changes, while the kidney had no evident damage. CONCLUSION: A eukaryotic expression vector pcDNA3-HBV containing HBV complete genome is constructed successfully. HepG2 cells transfected with the recombinant can express PreS1 mRNA and HBsAg stably. Specific cellular immune response can be induced in mice immunized by pcDNA3-HBV. A mice model of acute hepatitis with HBV has been established.

Antisense oligonucleotide targeting at the initiator of hTERT arrests growth of hepatoma cells.

AIM: To evaluate the inhibitory effect of antisense phosphorothioate oligonucleotide (asON) complementary to the initiator of human telomerase catalytic subunit (hTERT) on the growth of hepatoma cells. METHODS: The as-hTERT was synthesized by using a DNA synthesizer. HepG2.2.15 cells were treated with as-hTERT at the concentration of 10 micromol/L. After 72 h, these cells were obtained for detecting growth inhibition, telomerase activity using the methods of MTT, TRAP-PCR-ELISA, respectively. BALB/c(nu/nu) mice were injected HepG2.2.15 cells and a human-nude mice model was obtained. There were three groups for anti-tumor activity study. Once tumors were established, these animals in the first group were administered as-hTERT and saline. Apoptosis of tumor cells was detected by FCM. In the 2nd group, the animals were injected HepG2.2.15 cells together with as-hTERT. In the third group, the animals were given as-hTERT 24 hours postinjection of HepG2.2.15 cells. The anti-HBV effects were assayed with ELISA in vitro and in vivo. RESULTS: Growth inhibition was observed in cells treated with as-hTERT in vitro. A significant different in the value of A570-A630 was found between cells treated with as-hTERT and control (P<0.01) by MTT method. The telomerase activity of tumor cells treated with as-hTERT was reduced, the value of A450 nm was 0.42 compared to control (1.49) with TRAP-PCR-ELISA. The peak of apoptosis in tumor cells given as-hTERT was 21.12%, but not seen in saline-treated control. A prolonged period of carcinogenesis was observed in the second and third group animals. There was inhibitory effect on the expression of HBsAg and HBeAg in vivo and in vitro. CONCLUSION: As-hTERT has an anti-tumor activity, which may be useful for gene therapy of tumors.

[Effects of wide band frequency noise on NMDAR1(zeta 1), NMDAR2A(epsilon 1) subunit and ABR threshold in the different area of brain of AD rats poisoned by glutamic acid].

AIM: To investigate the change of NMDAR1 (zeta 1) subunit expression in temple cortex, frontal lobe, hippocampus and cerebellum of three different group rat after 98 dB wide frequency noise exposure. METHODS: Western Blot and RT-PCR technique, combined with auditory brainstem response (ABR) measurement. RESULTS: (1) Expressions of NMDAR1 (zeta 1) subunit in frontal cortex, temple cortex, hippocampus and cerebellum have no difference, but AD model rat is much weaker than the control group. (2) Expression of NMDAR2A (epsilon 1) in temple cortex for physiological saline groups rat have a mostly increase (plus noise), moreover, those are weakest expression in hippocampus. NMDAR1 (zeta 1) subunit in cerebellum have highest expression, moreover, it is weakest in temple cortex. (3) NMDAR1 (zeta1), NMDAR2A (epsilon 1) subunit expression in hippocampus for three groups rat have a down-regulation after adding noise. (4) NMDAR1 (zeta 1), NMDAR2A (epsilon 1) subunit mRNA expression in control group have no remarkable difference in different cortex. (5) Expressions of NMDAR2A (epsilon 1) in frontal temple cortex, hippocampus for AD model rat are less than that of other groups, weakest in cerebellum, weaker in frontal. CONCLUSION: Wide band frequency noise can reduce the expression of NMDAR1 (zeta 1) subunit in hippocampus and cerebellum of AD model rat, however, the way of regulation is not in the mRNA level. Wide band frequency noise can inhibit the expression of NMDAR2A (epsilon 1) in hippocampus, temple cortex of AD model rat, which has been regulated by mRNA level and have cortex area difference.

[Inhibition of HBV DNA replication and expression in 2.2.15 hepatoma cells infected with AFP-mediated HBX antisense RNA].

OBJECTIVE: To study the specific expression of the antisense RNA against hepatitis B virus X (HBX) gene in hepatoblastoma cell line and its anti -HBV activity. METHODS: HBX gene (nt.1370-1827) was amplified by PCR, then cloned into EB virus vector pEBAF which contained human alpha-fetoprotein promoter and enhancer. After transfected into 2.2.15 hepatoma cells and ECV304 human endothelial cells by lipofectin, northern blot, ELISA and real-time qualitative PCR were carried out to assay the expression of HBX mRNA, HBV antigens and HBV DNA level, respectively. RESULTS: The HBX antisense RNA expression vector pEBAF-as-HBX which could be expressed specifically in 2.2.15 hepatoblastoma cells was successfully constructed. Both HBV DNA level and the expressions of hepatitis B virus surface antigen (HBsAg) and e antigen (HBeAg) in 2.2.15 hepatoblastoma cells were inhibited by pEBAF-as-HBX. Compared with those in sense control (pEBAF-s-HBX), the inhibitory rates of HBsAg, HBeAg, and HBV DNA were 37.9%, 36.8%, and 25%, respectively. CONCLUSIONS: The pEBAF-as-HBX expression vector may lead to targeted-expression of HBX antisense RNA in hepatoma cells and shows great inhibition effect on HBV.