A novel dual-luciferase assay for anti-HIV drug screening based on the CCR5/CXCR4 promoters.

Acquired immunodeficiency syndrome (AIDS) is a serious worldwide disease caused by infection with the human immunodeficiency virus (HIV). C-C chemokine receptor 5 (CCR5) and C-X-C chemokine receptor 4 (CXCR4) are important coreceptors mediating HIV-1 cell entry. Many new anti-HIV drugs are currently in preclinical and clinical trials; however, drug development has proceeded slowly partly because of the lack of a high-throughput system to screen these drugs. Here, we describe the development of a novel dual-luciferase assay using a CCR5/CXCR4 promoter-driven firefly and Renilla luciferase vector (pGL4.10-RLUC-CCR5/CXCR4). Drugs were screened for the ability to regulate CCR5 and CXCR4 promoter activities. The CCR5 and CXCR4 promoters were inserted separately into the recombinant vector and transfected into the acute T lymphocyte leukemia cell line H9. Treatment of stable transfected cells with four traditional Chinese medicine compounds resulted in the dose-dependent inhibition of the CXCR4 and CCR5 promoter activities. The dual-luciferase reporter assay provides a rapid and direct method to screen anti-AIDS/HIV drugs.

Chinese materia medica used in medicinal diets.

ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal diets have a history of more than 2000 years. Locally referred to as yaoshan (Chinese: ), a medicinal diet is understood in China as a dietary product that combines herbs and food with the purpose of preventing and treating diseases or improving health under the guidance of traditional Chinese medicine theory. Medicinal diets are used in Chinese people's daily life and in specialized restaurants. Hundreds of Chinese materia medica (CMM) are used in medicinal diets; however, a comprehensive evaluation of medicinal diets is lacking. AIMS OF THE STUDY: This is an exploratory study that aims to identify the CMM that are most frequently used in medicinal diets and to provide an updated view of the current situation of medicinal diets in China. MATERIALS AND METHODS: A field study of 1221 people in 32 Chinese provinces was conducted over a period of approximately 6 months and included various types of interviews as well as a written questionnaire. Two approaches were used to analyse the data collected in the survey: (1) estimating the frequency of CMM consumed in daily diets; and (2) collecting CMM used in medicinal diet restaurants. Complementary information on the selected CMM was obtained from relevant databases, including PubMed, Google Scholar, Baidu Scholar, CNKI, and Web of Science. RESULTS: Ten CMM were reported as commonly used by more than 50% of the participants. Among these 10 species, most medicinally used parts were seeds and fruits. Pharmacological data from the literature revealed that these species are associated with a wide spectrum of biological properties, including antitumour (80%), antioxidant (50%), anti-diabetic (40%), antilipemic (40%), anti-aging (40%), antimicrobial (40%) and cardioprotective (40%) activities. Our survey shows that most medicinal diet restaurants are located in the eastern part of China, with the greatest numbers being found in Beijing and Guangzhou. Only Dioscoreae Rhizoma, Lycii Fructus, Chrysanthemi Flos and Longan Arillus were frequently consumed both in daily diets and at medicinal diet restaurants. Some of the similarities shared by these 4 species include an extensive history of use (>2000 years); a sweet flavour; and antioxidant, antidiabetic, antilipemic and cardioprotective effects. CONCLUSIONS: The 10 most commonly consumed CMM possess various biological effects that are currently target the most frequent health problems for the majority of the population. The development of medicinal diet restaurants has certain regional restrictions and is associated with the local climate environment and dietary culture. The data revealed by this study provided useful information for commercial exploitation of medicinal diets and their components and serve as a basis for further studies on various aspects of medicinal diets.

Investigation of free amino acid, total phenolics, antioxidant activity and purine alkaloids to assess the health properties of non-Camellia tea.

To find novel functional beverages from folk teas, 33 species of frequently used non-Camellia tea (plants other than Camellia) were collected and compared with Camellia tea (green tea, pu-erh tea and black tea) for the first time. Data are reported here on the quantities of 20 free amino acids (FAAs) and three purine alkaloids (measured by UHPLC), total polyphenols (measured by Folin-Ciocalteu assay), and antioxidant activity (DPPH). The total amounts of FAAs in non-Camellia tea (0.62-18.99 mg/g) are generally less than that of Camellia tea (16.55-24.99 mg/g). However, for certain FAAs, the quantities were much higher in some non-Camellia teas, such as γ-aminobutyric acid in teas from Ampelopsis grossedentata, Isodon serra and Hibiscus sabdariffa. Interestingly, theanine was detected in tea from Potentilla fruticosa (1.16±0.81 mg/g). Furthermore, the content of polyphenols in teas from A. grossedentata, Acer tataricum subsp. ginnala are significantly higher than those from Camellia tea; teas from I. serra, Pistacia chinensis and A. tataricum subsp. ginnala have remarkable antioxidant activities similar to the activities from green tea (44.23 µg/mL). Purine alkaloids (caffeine, theobromine and theophylline) were not detected in non-Camellia teas. The investigation suggest some non-Camellia teas may be great functional natural products with potential for prevention of chronic diseases and aging, by providing with abundant polyphenols, antioxidants and specific FAAs.

A potential in vitro and in vivo anti-HIV drug screening system for Chinese herbal medicines.

Chinese herbal medicines are often applied as an alternative therapy for viral diseases. However, the development of anti-HIV herbal drugs has proceeded slowly, partly because of the lack of a high-throughput system for screening these drugs. The present study evaluated 16 herbal medicines for anti-HIV activities in vitro and in vivo. Herbal medicines were first screened for the ability to regulate C-X-C receptor 4 (CXCR4) and C-C receptor 5 (CCR5) promoter activities. A single-round pseudotyped HIV-luciferase reporter virus system (HIV-Luc) was used to identify potential anti-HIV mechanisms. CD4+ T cells from healthy volunteers were examined for changes in CXCR4 and CCR5 levels. HIV-1 replication was evaluated by ELISA. Spica Prunellae and Herba Andrographitis were found to down-regulate the activities of both the CXCR4 and CCR5 promoters. Also, Spica Prunellae and Herba Andrographitis (>1000 µM) inhibited HIV-1 in a dose-dependent manner. CXCR4 and CCR5 levels were reduced in CD4+ T cells from healthy volunteers (p<0.05). Spica Prunellae and Herba Andrographitis (EC50: 3.18 and 5.49 µg/mL, respectively) could suppress cell fusion and decrease p24 antigen. In conclusion, the data demonstrated that Spica Prunellae and Herba Andrographitis possessed anti-HIV-1 capabilities, perhaps through the inhibition of the CXCR4 and CCR5 promoters and HIV-1 replication.

[Effect and mechanism of Andrographitis Herba on human CD4+ T cell promoters CXCR4 and CCR5].

OBJECTIVE: Utilizing a gene reporter technique to study the effects of Andrographitis Herba on human CXCR4 and CCR5 promoters. METHOD: Inhibition of CXCR4 and CCR5 on T cells of healthy volunteers was analyzed by RT PCR, Western blot and flow cytometry. The human CXCR4 and CCR5 promoters driving a luciferase reporter in vectors pGLA. 17-CXCR4 and pGLA. 17-CCR5 were transfected into H9 stem cells. G418 was used for selecting stable cell lines. Rat sera thus medicated was collected and added to the transfected H9 cells, in which the expression of CXCR4 and CCR5 promoters was detected. RESULT: They showed that the mRNA and protein expression levels of CXCR4 and CCR5 in human CD4+ T cells decreased significantly after taking Andrographitis Herba (P<0.05). Furthermore human CXCR4 and CCR5 promoter activity was downregulated significantly by sera from rats medicated with Andrographitis Herba. CONCLUSION: Andrographitis Herba may have the effect of down-regulating CXCR4 and CCR5 promoters. It provides a feasible experimental platform for screening herbal medicine as the treatment of HIV/AIDS.

[Establishment and initial application of a medicine screening technique based on human promoter of CCR5].

This research ws carried out to construct a medicine screening system targeting at human promoter of CCR5. The gene Human promoter of CCR5 was inserted into the rebuilt vector pGL3-neo. The pGL3-neo-CCR5 plasmids were transfected into Jurkat cells (the cell line of acute T lymphocyte leukemia). The lasting transfected cells were screened by G418. After seven kinds of traditional Chinese medicine had acted separately on the lasting transfected cells for 16h, the expression levels of CCR5 promoter in the cells were detected. The results showed that the level of luciferase activity of Shuanghuanglian-injectio group was remarkably lower than that of control (P < 0.05), and the levels of luciferase activity of Chuanhuning group, Baical skullcap root group, and Milkvetch root group were remarkably higher than that of control (P < 0.01). Shuanghuanglian-injectio depressed the activity of the transfected CCR5 promoter in cells cultivated in vitro; Chuanhuning, Baical skullcap root and Milkvetch root boosted the activity of the transfected CCR5 promoter in cells cultivated in vitro. Thus a medicine screening system based on Human promoter of CCR5 was initially constructed.