Cajanolactone A, a stilbenoid from Cajanus cajan, inhibits energy intake and lipid synthesis/storage, and promotes energy expenditure in ovariectomized mice.

BACKGROUND: We had reported that cajanolactone A (CLA) from Cajanus cajan dose-dependently inhibited ovariectomy-induced obesity and liver steatosis in mice, showing potential to prevent postmenopausal obesity and fatty liver. In this study, the role of CLA in the regulation of energy and lipid homeostasis was investigated. METHODS: Ovariectomized mice treated with CLA or vehicle for 12 weeks were performed a 48 h monitoring for energy metabolism and food uptake. After that, hypothalami, perigonadal (pWATs), inguinal (iWATs) and brown (BATs) adipose tissues, livers, sera, and fecal and cecal contents were collected and analyzed. FINDINGS: In CLA-treated mice, we observed reduced food uptake; increased energy expenditure; inhibited expression of orexigenic genes (ORX, ORXR2, pMCH and Gal) in the hypothalami, of lipogenic genes (CD36, SREBP-1c, ChREBP, PPARγ) in the livers, and of lipid storage proteins in the WATs (FSP27, MEST and caveolin-1) and livers (FSP27, Plin2 and Plin5); stimulated expression of metabolism-related proteins (pATGL and Echs1) in the adipose tissues and of thermogenic protein (UCP1) in the inguinal WATs; increased BAT content; increased mitochondria in the pWATs and livers; inhibited angiogenesis in the pWATs; and altered gut microbiome diversity with an increased abundance of Bacteroides. INTERPRETATION: CLA prevents ovariectomy-induced obesity and liver steatosis via regulating energy intake and lipid synthesis/storage, promoting UCP1-dependent heat production, and protecting the mitochondrial function of hepatocytes and adipocytes. The improved gut microecology and inhibited angiogenesis may also contribute to the anti-obese activity of CLA.

Cajanolactone A, a stilbenoid from cajanus cajan, prevents ovariectomy-induced obesity and liver steatosis in mice fed a regular diet.

BACKGROUND: Visceral obesity and fatty liver are prevalent in postmenopausal women. The stilbene-rich extract of Cajanus cajan (L.) Millsp. has been reported to prevent ovariectomy-induced and diet-induced weight gain in animal models, and stilbenoids from C. cajan are thought to have the potential to prevent postmenopausal obesity and fatty liver. PURPOSE: Cajanolactone A (CLA) is the main stilbenoid from C. cajan with osteoblastogenic promoting activity. This study investigated the potential of CLA to prevent postmenopausal obesity and fatty liver. Underlying mechanisms were also investigated. METHOD: Ovariectomized C57BL/6 mice fed a regular diet were used as mimics of postmenopausal women and given 10, 20, or 40 mg/kg/d of CLA, 0.1 mg/kg/d of estradiol valerate (EV, positive control), or vehicle (OVX) orally for 16 weeks. Mice of the same age subjected to a sham operation were used as control (Sham). Body weights were recorded every 2 weeks for 16 weeks. Body compositions were analyzed via micro-CT. Serum levels of lipids, adipocytokines and aminotransferases were measured using the relevant kits. mRNA levels of genes of interest were detected by RT-qPCR. Proteomic study of perigonadal white adipose tissue (pWAT) was performed using tandem-mass-tags-based proteomic technology combined with Parallel-Reaction-Monitoring (PRM) validation. RESULTS: CLA showed potential equivalent to that of EV to prevent ovariectomy-induced overweight, obesity, dyslipidemia, liver steatosis and liver dysfunction, but did not prevent uterine atrophy. In the liver, CLA significantly inhibited ovariectomy-induced upregulation in expression of lipogenic genes SREBP-1c and ChREBP, and stimulated the mRNA expression of apolipoprotein B gene ApoB. In pWAT, CLA reversed, or partially reversed ovariectomy-induced downregulation in the expression of a number of metabolism- and mitochondrial-function-related proteins, including Ndufa3, Pcx, Pdhb, Acly, Acaca, Aldh2, Aacs and Echs1. In addition, ovariectomy-inhibited mRNA expression of Pdhb, Aacs, Acsm5, Echs1, and Aldh2 genes in pWAT was also reversed. CONCLUSION: CLA was demonstrated to be a potential non-estrogen-like drug candidate for prevention of postmenopausal obesity and fatty liver. The underlying mechanism might involve the inhibition of lipogenesis and promotion of triglycerides output in the liver, and the promotion of metabolism and mitochondrial functions of visceral white adipose tissue.

Cajanolactone A from Cajanus cajan Promoted Osteoblast Differentiation in Human Bone Marrow Mesenchymal Stem Cells via Stimulating Wnt/LRP5/ß-Catenin Signaling.

Cajanolactone A (CLA) is a stilbenoid discovered by us from Cajanus cajan (L.) Millsp. In our study, CLA was found to promote osteoblast differentiation in human bone marrow mesenchymal stem cells (hBMSCs), as judged by increased cellular alkaline phosphatase activity and extracellular calcium deposits, and elevated protein expression of Runx2, collagen-1, bone morphogenetic protein-2, and osteopontin. Mechanistic studies revealed that hBMSCs undergoing osteoblast differentiation expressed upregulated mRNA levels of Wnt3a, Wnt10b, LRP5/6, Frizzled 4, ß-catenin, Runx2, and Osterix from the early stage of differentiation, indicating the role of activated Wnt/ß-catenin signaling pathway in osteoblast differentiation. Addition of CLA to the differentiation medium further increased the mRNA level of Wnt3a, Wnt10b, Frizzled 4, LRP5, and ß-catenin, inferring that CLA worked by stimulating Wnt/LRP5/ß-catenin signaling. Wnt inhibitor dickkopf-1 antagonized CLA-promoted osteoblastogenesis, indicating that CLA did not target the downstream of canonical Wnt signaling pathway. Treatment with CLA caused no changes in mRNA expression level, as well as protein secretion of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL), indicating that CLA did not affect the OPG/RANKL axis. Our results showed that CLA, which promoted osteoblast differentiation in hBMSCs, through activating Wnt/LRP5/ß-catenin signaling transduction, is a promising anti-osteoporotic drug candidate.

[Immune regulation effect of Chinese medicine of invigorating spleen and kidney detoxification on simian immunodeficiency virus-infected rhesus macaque].

To evaluate the effect of Chinese medicine of invigorating spleen and kidney detoxification on simian immunodeficiency virus-infected rhesus macaque. Eight SIV rhesus macaques of the same age were randomly divided into Chinese medicine of invigorating spleen and kidney detoxification group(hereinafter referred to as Chinese medicine group) and anti-virus drug(HAART) group. The traditional Chinese medicine and antiviral therapy were given for 8 weeks, and peripheral blood was collected for detection in every 4 weeks. The results showed that Chinese medicine of invigorating spleen and kidney detoxification could not obviously decrease plasma viral load as HAART, but it can increase CD4 number in peripheral blood, especially the CD4 naive cells, and increase the number of CD4 and CD8 cells, enhance the immune response to pathogens. Therefore, it delayed the occurrence and development of spleen deficiency to a certain extent, indicating that the medicine had immune regulation effect, with considerable clinical value and application prospects.

Combinations of 1,8-cineol and oseltamivir for the treatment of influenza virus A (H3N2) infection in mice.

It is need for development of new means against influenza virus due to the lack of efficacy of available therapeutic strategies. In previous research, 1,8-cineol exert its inhibition of nuclear factor (NF)-κB, the main regulator of cytokine and chemokine production in influenza, and anti-inflammatory activity. These fact supports and helps establish the hypothesis that 1,8-cineol may have synergism with an antiviral on influenza virus infection. The combined effect of 1,8-cineol with oseltamivir in a mouse type A influenza virus (Victoria/3/75,H3N2) model were examined. We initially tested combinations of 1,8-cineol (30, 60, and 120 mg/kg/day) and oseltamivir (0.1, 0.2, and 0.4 mg/kg/day). In addition, the 0.4 mg/kg/day of oseltamivir combined with 120 mg/kg of 1,8-cineol was selected for further combination studies. Oseltamivir was 30%, 40%, and 60% protective at 0.1, 0.2, and 0.4 mg/kg/d. Combinations of 1,8-cineol (30, 60, and 120 mg/kg/d) and oseltamivir (0.1, 0.2, and 0.4 mg/kg/d) increased the number of survivors and mean survival time (MST) following combination treatment was greater than monotherapy alone. Three dimensional analysis of drug interactions using the MacSynergy method showed a strong synergistic effect of these drug combinations. Survival, MST, lung parameters (lung index, viral titers, and pathology), and cytokines (IL-10, TNF-α, IL-1ß, and IFN-γ) expression in lung demonstrated the high effectiveness of the combination. Combined treatment was associated with longer MST and more reduced cytokine levels than oseltamivir alone. These data demonstrate that combinations of 1,8-cineol and oseltamivir have synergistic effect against influenza A virus (H3N2) infection.

Aikeqing decreases viral loads in SHIV89.6-infected Chinese rhesus macaques.

BACKGROUND: Aikeqing (AKQ) has been shown in clinical studies to improve quality of life of HIV/AIDS patients, but anti-HIV activity has not been determined. The SHIV-infected macaque is an important animal model for testing antiviral drugs. This study aimed to determine the anti-HIV activity of AKQ in chronically SHIV89.6-infected Chinese rhesus macaques. METHODS: Nine Chinese rhesus macaques were inoculated intravenously with SHIV89.6 virus. At 11 weeks post-infection, the animals were arbitrarily divided into three groups: high-dose (AKQ 1.65 g/kg; n = 3), low-dose (AKQ 0.55 g/kg; n = 3), and control (water 1 mL/kg; n = 3). Treatment was administered by the intragastric gavage route once-daily for 8 weeks. Blood (5 mL) was collected biweekly. Viral loads were analyzed by real-time quantitative RT-PCR assays, and T cell counts were monitored by FACS analyses throughout the treatment. RESULTS: AKQ induced a persistent decline (P = 0.02) in plasma viral loads during treatment in the high-dose group compared with their baseline levels, and cessation of the therapy caused viral load rebound to the pretreatment levels. No significant difference (P = 0.06) was found in the plasma viral loads during treatment in the low-dose group. The CD4(+) T cell counts and CD4/CD8 ratios remained at stable high levels during the treatment period. CONCLUSION: AKQ reduced plasma viral loads in the SHIV89.6-infected Chinese rhesus macaque model.

A Triterpenoid Inhibited Hormone-Induced Adipocyte Differentiation and Alleviated Dexamethasone-Induced Insulin Resistance in 3T3-L1 adipocytes.

6α-Hydroxylup-20(29)-en-3-on-28-oic acid (1), a natural triterpenoid, was found to possess the ability in a dose-dependent manner inhibiting hormone-induced adipocyte differentiation in 3T3-L1 preadipocytes, and restoring glucose consuming ability in dexamethasone (DXM)-induced insulin resistant 3T3-L1 adipocytes. Compound 1 was also found to ameliorate DXM-induced adipocyte dysfunction in lipolysis and adipokine secretion. Mechanistic studies revealed that 1 inhibited adipocyte differentiation in 3T3-L1 preadipocytes via down-regulating hormone-stimulated gene transcription of peroxisome proliferator-activated receptor γ and CCAAT-enhancer-binding protein alpha which are key factors in lipogenesis, and restored DXM-impaired glucose consuming ability in differentiated 3T3-L1 adipocytes via repairing insulin signaling pathway and activating down-stream signaling transduction by phosphorylation of signaling molecules PI3K/p85, Akt2 and AS160, thus leading to increased translocation of glucose transporter type 4 and transportation of glucose.

[Pharmacokinetic Effect of Aikeqing Granule by Different Medication Ways on Zidovudine in HAART of Rats].

OBJECTIVE: To study pharmacokinetic effect of Aikeqing Granule (AG) by different medication ways on zidovudine (AZT) in highly active antiretroviral therapy ( HAART) of rats. METHODS: Totally 36 rats were administered with corresponding medications by gastrogavage, group I [HAART: AZT 31.5 mg/kg +3TC 31.5 mg/kg + Efavirenz (EFV) 63.0 mg/kg], group II (HAART+AG525 mg/kg), group III (HAART and AG 525 mg/kg after a 2-h interval). Drug concentrations of AZT were determined by high performance liquid chromatography-mass spectroscopy (HPLC-MS) before HAART, and at 0.5, 1, 2, 3, 4, 6, 8, 10, 12 h after HAART, respectively. Pharmacokinetic parameters [such as t1/2, Tmax, Cmax, AUCo-t, plasma clearance rate (CL)] were calculated by DAS2.0 Software. RESULTS: The-equation of linear regression of AZT was good, with the precision, coefficient of recovery, and stability definitely confirmed. AUC in group II and III was larger than that of group I. There was no statistical difference in t1/2, Tmax, Cmax, AUC0-12 h, or AUC0-∞ among groups (P > 0.05). CONCLUSION: AG combined HAART could enhance the Cmax of AZT.

[Experimental observation of SIVmac239 Chinese rhesus monkey model at sub-acute phase of AIDS].

OBJECTIVE: To observe T lymphocyte subsets and indicators of changes in viral load in sub-acute period in Chinese rhesus monkey model of AIDS SIVmac239. To explore Virology related index variation in sub-acute period of the Chinese rhesus monkey model of AIDS. METHOD: To replicate Chinese rhesus monkey model of AIDS, healthy Chinese rhesus monkey was inoculated with SIVmac239 viral strain. To observe changes in T lymphocyte subsets indexes and viral load after infection with the simian immunodeficiency virus (SIV) in sub-acute period on an animal model. The clinical symptoms of the animal model was recorded simultaneously. RESULT: During the 10 weeks after SIV acute infection, body weight and BMI index were relatively stable, the difference was not significant at all time points. Twelve monkeys were tested SIV positive by real-time PCR after three days of infection. On the 7th day after infection, 15 monkeys were tested SIV positive. Viral load increased rapidly, but reached a peak on the 10th-14th day after infection, then showed a level of volatility decline. T lymphocyte subsets showed significant changes, among them, CD3% and CD3 counts fluctuated upward trend and reached to the highest level in two weeks after infection; of CD4% and CD4 count changes were not synchronized, CD4% declined trend while the CD4 count was an increasing trend after the infection; of CD8% and CD8 counts fluctuate upward trend, and reached to a highest level in two weeks after infection ;the ratio of CD4/CD8 and the counts of CD4CD28 T cells decreased significantly in two weeks after infection; the former followed by a slow decline, the latter followed by a rapid rise. Three mouths after the infection 3 monkeys showed significant clinical symptoms. One of the rhesus monkeys had symptoms of diarrhea and two of them had reduced food intake. CONCLUSION: This experiments established standardization of Chinese Rhesus monkeys used in the research of AIDS and provide a detailed contents in the changes of sub-acute phase.

[Study on the regional features of Chinese medicine etiology and pathogenesis of AIDS].

OBJECTIVE: To analyze the differences in the Chinese medicine (CM) etiology and pathogenesis of AIDS. METHODS: A cross-sectional investigation study of AIDS patients and non-infected people was carried out in Henan, Guangdong and Xinjiang provinces respectively by questionnaire of clinical epidemiology and analyzed with frequencies of symptoms and signs combined with syndrome factors. The distribution differences of syndrome factors in the 3 provinces were compared. RESULTS: Totally 321 cases were investigated in the 3 provinces. As for the syndrome factors of AIDS, qi deficiency, blood deficiency, yin deficiency, yang deficiency, dampness evil, phlegm, qi stagnation, essence deficiency, and so on were dominated in the 3 provinces, but with their own features. For example, the scores of dampness evil were higher in Guangdong Province. Yin deficiency was dominated in Xinjiang Province. The scores of Henan Province were generally higher. As for the location of syndrome factors, Shen, Fei, Pi, and Gan were dominated in the 3 provinces. The score of Pi was the highest in Guangdong Province, while the score of Shen was the highest in Henan and Xinjiang provinces. CONCLUSIONS: AIDS has regional features in the nature and the syndrome factors of location in different provinces. It is necessary to take into consideration the regional factors when analyzing the pathogenesis of AIDS.

[Traditional Chinese medicine syndrome factors of patients with HIV infection or AIDS in China].

OBJECTIVE: To study the characteristics of traditional Chinese medicine (TCM) syndrome factors of patients from different areas of China with human immunodeficiency virus (HIV) infection or acquired immunodeficiency syndrome (AIDS). METHODS: A cross-sectional investigation study was conducted in Henan, Guangdong and Yunnan Provinces and Xinjiang Uygur Autonomous Region of China from October 2008 to August 2010. Based on literature review and expert opinion, a clinical questionnaire of TCM syndromes was drawn up. This survey was carried out after the investigators were professionally trained. Wenfeng III Auxiliary Diagnosis and Treat System of TCM was used to analyze the frequencies of AIDS patients' signs and symptoms with scores above 70 of syndrome factors respectively. Based on this work, syndrome factors of AIDS were analyzed in different areas. RESULTS: There were 608 HIV/AIDS cases investigated from October 2008 to August 2010 in total; among them, 276 cases were from Henan, 126 cases from Guangdong, 120 cases from Xinjiang and 86 cases from Yunnan. The results of syndrome factor analysis indicated that the syndromes of four provinces were similar. HIV/AIDS patients in the four areas exhibited qi deficiency, blood deficiency, yin deficiency, yang deficiency, dampness, phlegm, qi stagnation and essence deficiency syndromes. Patients in each area also had their own characteristics, such as that the scores of dampness of Guangdong and yin deficiency of Xinjiang were higher than the other syndromes, whereas the scores of Henan Province were higher than the other areas. AIDS patients had higher scores of syndromes than HIV-infected patients. CONCLUSION: HIV/AIDS patients from different areas had similar syndrome elements. The theory of "AIDS toxin injuring primordial qi" can sum up the TCM etiology and pathogenesis of HIV/AIDS.

Dihydroartemisinin upregulates death receptor 5 expression and cooperates with TRAIL to induce apoptosis in human prostate cancer cells.

Dihydroartemisinin (DHA) is a derivative of artemisinin and is an effective anti-malaria therapeutic used worldwide. In this paper, we report that DHA is as a potential anticancer drug for prostate cancer. Our data indicate that DHA suppresses the PI3-K/Akt and ERK cell survival pathways and triggers the induction of death receptor DR5 and activation of extrinsic and intrinsic cell death signaling. DHA-mediated DR5 induction appears to occur via increased transcriptional activity of DR5 promoter. Our data also show that, while DHA has strong cytotoxicity in tumor cells, it exhibits minimal cytotoxic effects on normal prostate epithelial cells. Our studies also demonstrate that DHA worked cooperatively with death ligand TRAIL. Combination of DHA and TRAIL significantly enhanced cell killing above that noted with a single agent alone. Based on these results, we propose a novel idea of developing DHA alone and/or in combination with TRAIL for the treatment of prostate cancer.

A new 3-benzylchroman derivative from Sappan Lignum (Caesalpinia sappan).

3'-Deoxy-4-O-methylepisappanol, a new 3-benzylchroman derivative, was isolated from Sappan Lignum, together with thirteen known chemical compounds identified as protosappanin A, sappanchalcone, sappanone B, palmitic acid, (+)-(8S,8'S)-bisdihydrosiringenin, brazilein, 3-deoxysappanchalcone, (+)-lyoniresinol, 3-deoxysappanone B, protosappanin B, isoprotosappanin B, 3'-O-methylbrazilin and brazilin, respectively. Among these known compounds, this is the first time that (+)-(8S,8'S)-bisdihydrosiringenin was obtained from the family Caesalpiniaceae.

Tenacigenin B derivatives reverse P-glycoprotein-mediated multidrug resistance inHepG2/Dox cells.

Tenacissimoside A (1) and 11alpha-O-benzoyl-12beta- O-acetyltenacigenin B (2), two derivatives of tenacigenin B (3) from the plant Marsdenia tenacissima, reversed multidrug resistance in P-glycoprotein (Pgp)-overexpressing multidrug-resistant cancer cells. The sensitivity of HepG2/Dox cells to the antitumor drugs doxorubicin, vinblastine, puromycin, and paclitexel was increased by 18-, 10-, 11-, and 6-fold by 20 microg/mL (or 25 microM) of 1 and 16-, 53-, 16-, and 326-fold by 20 microg/mL (or 39 microM) of 2, respectively. A preliminary mechanistic study has suggested that 1 might modulate Pgp-mediated multidrug resistance through directly interacting with the Pgp substrate site.

[Artesunate in interrupting the transmission of Plasmodium falciparum].

OBJECTIVE: To observe the effect of artesunate (ATS) on the infectivity of Plasmodium falciparum gametocytes (PFG). METHODS: 31 volunteers with falciparum malaria and gametocytaemia were randomly divided into 3 groups: artesunate (ATS) group (15 cases), quinine (QN) group (10 cases) and placebo group (6 cases). Each case in ATS group received 6-day course of oral artesunate (200 mg at 0, 6 and 24 hours then 100 mg daily for 4 days). Cases in QN group each received 21-dose course of quinine sulfate (500 mg/time) over seven days. Cases in placebo group took 2 tablets of vitamin B composites, three times per day for seven days. Peripheral PFG were counted daily in all cases until the clearance of PFG. Mosquitoes (Anopheles dirus) were fed with venous blood of patients on the 1st, 7th, 14th, 21st and 28th day, respectively. RESULTS: All cases in placebo group were PFG positive at the whole course by blood smear examinations. The PFG relative density in ATS group were (12.5+/-3.3)%, (1.2+/-0.4)%, (0.3+/-0.1)% on 7th, 14th, 21st day respectively, and the mean PFG clearance time was (22.0+/-1.4) d. The PFG relative density in QN group were (173.9+/-47.0)%, (112.5+/-45.4)%, (32.5+/-17.8)% at 7th, 14th, 21st day respectively, and the mean clearance time of PFG was (32.5+/-2.1) d (t=4.731, P<0.01). PFG remained positive on the 28th day in placebo group. The infectivity test to mosquitoes showed on 14th day the positive rate in ATS group, QN group and placebo group were 0, 35.0% and 48.7% respectively. In ATS group, the sporozoite rate of anopheline mosquitoes were 14.8% and 0 at 7th, 14th day, while in QN group, 142.0%, 98.6% and 20.3% at 7th, 14th, 21st day respectively. In placebo group, the infection rate of sporozoites remained stable. CONCLUSION: Oral administration of artesunate with a total dosage of 1000 mg in 6 days inhibits the infectivity of PFG.

[Autoantibodies and autoimmunity in simian immunodeficiency virus-infected monkeys].

OBJECTIVE: To study the relationship between simian acquired immunodeficiency syndromn (SAIDS) and autoimmunity in simian immunodeficiency virus (SIV)-infected monkeys. METHODS: Indirect immunofluorescence assays were performed to detect plasma or serum autoantibodies in SIV-infected monkeys. The heart, liver, spleen, lung, kidney, and lymph node of BALB/c mice, a strain of endothelial cell ECV304, and granulocytes were used as target antigens. These results were compared with HE stained slides of SIV-infected monkeys. RESULTS: The levels of various autoantibodies, including anti-lymphocyte autoantibodies, anti-endothelial cell autoantibodies, and anti-granulocyte antibodies, increased after SIV infection in monkeys. Moreover, pathological examinations showed injuries in the lymphoid tissue and vascular pathological changes in cerebral cortex, submucosa of gastrointestinal tract, interstitial capillaries of myocardium, nephron of the kidney, and sinusoid cell of liver. CONCLUSION: The increased autoantibodies and the pathological changes of tissues and organs confirm the existence of autoimmunity in SIV-infected monkeys.

[Blood lipid-regulation of stilbene glycoside from polygonum multiflorum].

OBJECTIVE: To study lipid-regulating action of 2, 3, 5, 4'-tetrahydroxy stilbene-2-O-beta-D-glucopyranoside (TSG) from Polygonum multiflorum on experimental model hyperlipidemic rats. METHOD: TSG 90 and 180 mg x kg(-10 x d(-1), atorvastatin mg kg(-1) x d(-1) and saline 2 mL x d(-1) were administered to hyperlipidemic rats. Groups of rats were determined and compared with those of saline group. The LDLR and HMGR mRNA expression were also detected. RESULT: TSG significantly reduced serum TC and LDL-C level and atherosclerosis index, increased the expression of LDLR in the liver cells. CONCLUSION: TSG, which shows effects and mechanism in part like atorcastatin, is a major constituent with blood-lipid regulating effect of P. multiflorum and can be explored as a potent medication for hyperlipidemia. Effects on LDL-C and AI, as well as on gene expression of TSG were first reported.

Anti-HBV effect of liposome-encapsulated matrine in vitro and in vivo.

AIM: To study the anti-HBV effect of liposome-encapsulated matrine (Lip-M) in vitro and in vivo. METHODS: 2.2.15 cell line was cultured in vitro to observe the effect of Lip-M and matrine on the secretion of HBsAg and HBeAg. The toxicity of Lip-M and matrine to 2.2.15 cell line was also studied by MTT method. In in vivo study, drug treatment experiment was carried out on the 13th day after ducks were infected with duck hepatitis B virus (DHBV). The ducks were randomly divided into 4 groups with 5-6 ducks in each group. Lip-M and matrine were given to DHBV-infected ducks respectively by gastric perfusion. Four groups were observed: group of Lip-M (20 mg/kg), group of Lip-M (10 mg/kg), group of matrine (20 mg/kg) and group of blank model. The drug was given once daily for 20 d continuously, and normal saline was used as control. The blood was drawn from the posterior tibial vein of all ducks before treatment (T(0)), after the medication for 5 (T5), 10 (T10), 15 (T15), 20 (T20) d and withdrawl of the drug for 3 d (P3). The serum samples were separated and stored at -70 degrees, DHBV-DNA was detected by the dot-blot hybridization. RESULTS: After addition of Lip-M and matrine to 2.2.15 cell line for eleven d, the median toxic concentration (TC50) of Lip-M and matrine was 7.29 mg/mL and 1.33 mg/mL respectively. The median concentration (IC50) of Lip-M to inhibit HBsAg and HBeAg expression was 0.078 mg/mL and 3.35 mg/mL respectively. The treatment index (TI) value of Lip-M for HBsAg and HBeAg was 93.46 and 2.17 respectively, better than that of matrine. The DHBV-infected duck model treatment test showed that the duck serum DHBV-DNA levels were markedly reduced in the group of Lip-M (20 mg/kg) after treated by gastric perfusion for 10, 15 and 20 d (0.43+/-0.22 vs 0.95+/-0.18, t = 4.70, P = 0.001<0.01.0.40+/-0.12 vs 0.95+/-0.18, t = 6.34, P = 0.000<0.01. 0.22+/-0.10 vs 0.95+/-0.18, t = 8.30, P = 0.000<0.01), compared to the group of matrine (20 mg/kg) (0.43+/-0.22 vs 0.79+/-0.19, t = 3.17, P = 0.01<0.05. 0.40+/-0.12 vs 0.73+/-0.24, t = 3.21, P = 0.009<0.05. 0.22+/-0.10 vs 0.55+/-0.32, t = 2.27, P = 0.046<0.05.), and the control(0.43+/-0.22 vs 0.98+/-0.29, t = 3.68, P = 0.005<0.01. 0.40+/-0.12 vs 0.97+/-0.30, t = 4.26, P = 0.002<0.01. 0.22+/-0.10 vs 0.95+/-0.27, t = 5.76, P = 0.000<0.01). After the treatment for 20 d and withdrawl of the drug for 3 d, duck serum DHBV-DNA level in the group of Lip-M (10 mg/kg) markedly reduced (0.56+/-0.26 vs 0.95+/-0.38, t = 5.26, P = 0.003<0.05. 0.55+/-0.25 vs 0.95+/-0.38, t = 5.52, P = 0.003<0.05), and the difference was significant as compared with the control (0.56+/-0.26 vs 0.95+/-0.27, t = 2.37, P = 0.042<0.05. 0.55+/-0.25 vs 0.89+/-0.18, t = 2.55, P = 0.031<0.05), but not significant as compared with the group of matrine (20 mg/kg). After withdrawl of the drug for 3 d, the levels of DHBV-DNA did not relapse in both groups of Lip-M. CONCLUSION: Lip-M can evidently inhibit the replication of hepatitis B virus in vitro and in vivo; its anti-HBV effect is better than that of matrine.

Evaluation on expression of the recombinant S gene of human hepatitis B virus in vitro and in vivo.

OBJECTIVES: To construct a DNA vaccine capable of expressing the S gene of hepatitis B virus (HBV) and evaluate the expression of the recombinant S gene in vitro and in vivo. METHODS: A cloned S-X gene fragment was inserted into an eukaryote expression vector to construct a recombinant expressing plasmid pCMV-SX. The recombinant plasmid was transcribed in vitro with a T7 promoter transcription system and transfected into a human hepatoblastoma cell line HepG2. The expression of the S gene was detected by Northern blot hybridization, Western blot hybridization, and enzyme-linked immunosorbent assay (ELISA), respectively. BALB/c mice were inoculated with the recombinant plasmid, and the efficiency of DNA-based immunization in eliciting anti-HBs was evaluated by ELISA. RESULTS: In vitro transcription of the subcloned HBV S gene was confirmed by Northern blot hybridization. The results of Western blot hybridization and ELISA showed that the S gene was expressed exactly in HepG2. In immune experiment, 2 of 10 immunized mice were shown to induce antibody against HBsAg. CONCLUSION: The recombination and expression of the S gene can be achieved successfully in vitro. And the recombinant plasmid is able to elicit humoral immune response in mice.