Screening experiments for antiinflammatory properties

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This chapter describes some examples of screening experiments aimed at identifying antiinflammatory constituents of plants. A large number of plants and herbs are known for their anti-inflammatory properties. Well-known examples are willow bark (contains salicin, from which aspirin is derived), Boswellia serrata (boswellic acids) and turmeric (curcumin). In addition to these, many other herbs have been suggested to be anti-inflammatory. Inflammation plays a role in many different clinical disorders. In addition to the obvious inflammatory diseases such as arthritis, asthma, Crohn's disease, psoriasis and so on, inflammation also plays an important role in diseases such as atherosclerosis, diabetes, Alzheimer's and many other diseases. In many of these, a disordered immune system contributes to the onset and/ or progression of the disease.

4.6.1 Single target screening

What are important targets for anti-inflammatory therapies? A key regulatory factor in the inflammatory response is the transcription factor family NF-kB. This family of proteins is present in almost all human cells. In inflammatory cells such as macrophages and lymphocytes, NF-kB is activated after stimulation of the cells by a pro-inflammatory stimulus. Its activation leads to the transcription of many different genes involved in the inflammatory response, including cytokines such as TNFa. Inhibition of NF-kB attenuates the inflammatory response, and therefore it is a major target for the development of antiinflammatory drugs. An example of a relatively simple cell-based assay to screen for NF-kB activation is given in Fig. 4.1. The assay is a reporter gene assay, in which a total of five NF-kB elements are placed in front of the reporter gene. Thus, the reporter gene is expressed only when NF-kB is activated. The reporter gene plasmid is transfected into a macrophage-like cell line, in this case the mouse cell line RAW264.7. The cells are incubated with a stimulus that activates NF-kB, and in the absence of an inhibitor a strong reporter gene response is detectable. When cells are incubated with a stimulus together with an inhibitor, the reporter gene response is attenuated. In this way, possible inhibitory compounds present in plant-derived extracts may be detected. Experiments such as these always include controls for cell viability, to check for possible interference of toxic compounds present in extracts that may give rise to false positives. Obviously, positive and negative controls for activation and inhibition (i.e. a known inhibitor such as dexamethasone) of NF-kB are always included.

There are many other important targets for anti-inflammatory therapies, in addition to transcription factors. An important class of molecules are the receptors, for example receptors for cytokines, chemokines and eicosanoids (Onuffer and Horak, 2002; Holgate

Antiinflammatory Foods

Fig. 4.1 Example of a reporter gene assay to monitor inflammatory responses. One of the main events in the pro-inflammatory response of macrophages is the activation of the transcription factor NF-kB. This cell-based assay is designed to monitor this process. The basis is formed by a macrophage-like cell line that was stably transfected with a reporter gene (luciferase) under the control of a promoter that contains NF-kB responsive elements (RE). When these cells are exposed to proinflammatory substances (e.g. LPS), this will lead to signal transduction from the receptor into the cell towards NF-kB, which in turn is activated and induces the transcription of luciferase. The expression of the luciferase protein is monitored by measuring the luciferase activity using luminometry. The inset shows an example of an anti-inflammatory inhibitor that strongly reduces luciferase activity even in the presence of LPS, whereas the solvent control does not have an effect. This system is useful to screen for anti-inflammatory plant-derived substances that target the NF-kB pathway.

Fig. 4.1 Example of a reporter gene assay to monitor inflammatory responses. One of the main events in the pro-inflammatory response of macrophages is the activation of the transcription factor NF-kB. This cell-based assay is designed to monitor this process. The basis is formed by a macrophage-like cell line that was stably transfected with a reporter gene (luciferase) under the control of a promoter that contains NF-kB responsive elements (RE). When these cells are exposed to proinflammatory substances (e.g. LPS), this will lead to signal transduction from the receptor into the cell towards NF-kB, which in turn is activated and induces the transcription of luciferase. The expression of the luciferase protein is monitored by measuring the luciferase activity using luminometry. The inset shows an example of an anti-inflammatory inhibitor that strongly reduces luciferase activity even in the presence of LPS, whereas the solvent control does not have an effect. This system is useful to screen for anti-inflammatory plant-derived substances that target the NF-kB pathway.

et al., 2003). Adhesion molecules that are involved in the translocation of immune cells from the circulation into the sites of inflammation are also promising drug targets (Yusuf-Makagiansar et al., 2002). Other important targets are the signalling molecules themselves, such as cytokines and chemokines. The best-known example of this is TNFa: therapies based on the specific inhibition of TNFa have proven to be very efficacious for Crohn's disease and rheumatoid arthritis (Elliot et al., 1994; Targan et al., 1997).

4.6.2 Genomics-based screening

A much more complex experiment to test whether plant-derived extracts possess antiinflammatory activity is genomics-based screening. The basic principle is that, instead of just one or two targets being screened, the whole transcriptome, proteome or metabolome is analysed. As in 'normal' screening, an in vitro model system is usually used. This can be a macrophage, lymphocyte or any other type of relevant cell line, but it can also be applied to whole blood. The advantage of cell lines is that they provide a relatively stable background in which the experiments are performed.

In the example described here, a macrophage-like cell line was used. The cell line, U937 (Ralph et al., 1976), can be cultured in such a manner that it adopts a macrophage-like phenotype. This can be verified using macrophage-specific markers: in our case we

Macrophages

'Omics'

Macrophages

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