Chapter 3. Results
3.15 Two polyacetylenic compounds, identified from butanol
Our data showed that a butanol fraction of B. pilosa promoted that of Th0 into Th2 cells but inhibited the differentiation of Th0 into Th1 cells using FACS analysis.
We next tracked down and characterized the bioactive compounds with the efficacy on modulating T cell differentiation based on a BGFI principle. Here, two polyacetylenic compounds, 2-ß-D-glucopyranosyloxy-1-hydroxy-5(E)-tridecene- 7,9,11-triyne (1) and 3-ß-D-glucopyranosyloxy-1-hydroxy-6(E)-tetradecene-8,10,12- triyne (2), were identified as bioactive constituents that capable of modulating T cell differentiation. Our data showed that the compound (2) at 5, 10 and 15 μg/ml increased the percentage of IL-4-producing cells (i.e., Th2 cells) from 11% to 15%
(Figure 22A) whereas it at the same doses decreased that of IFN-γ-producing cells (i.e., Th1 cells) from 60% to 37% (Figure 22B). However, 2-ß-D-glucopyranosyloxy-1-hydroxy-5(E)-tridecene-7,9,11-triyne (1) showed, if any, only little inhibition (10%) of the differentiation of Th0 into Th1 cells and slight enhancement (8%) of the differentiation of Th0 into Th2 cells at the dose of 15 μg/ml (Figure 23). Taken together, our results display that this T cell differentiation method can be used as a highly efficient platform to distinguish bioactive phytochemicals from the crude extracts and lead to identification of bioactive phytocompounds.
Figure 22. The effect of 3-ß-D-glucopyranosyloxy-1-hydroxy-6(E)-tetradecene- 8,10,12-triyne (2) in T helper cell differentiation. (A) Human CD4+ T cells were cultured under Th2 condition in the presence of 3-ß-D-glucopyranosyloxy-1-hydroxy-6(E)-tetradecene-8,10,12-triyne (2) at 0, 5, 10 and 15 μg/ml. T cells were analyzed using FACS and the percentage of IL-4-producing cells was calculated. (B) Following the same treatment as described for Th1 cell differentiation (Figure 20), the percentage of IFN-γ-producing cells was calculated.
Data are representative of three experiments.
Figure 23. The effect of 2-ß-D-glucopyranosyloxy-1-hydroxy-5(E)-tridecene- 7,9,11-triyne (1) in T helper cell differentiation. (A) Human CD4+ T cells were cultured under Th2 condition in the presence of 2-ß-D-glucopyranosyloxy-1-hydroxy-5(E)-tridecene-7,9,11-triyne (1) at 0, 5, 10 and 15 μg/ml. T cells were analyzed using FACS and the percentage of IL-4-producing cells was calculated. (B) Following the same treatment as described for Th1 cell differentiation (Figure 20), the percentage of IFN-γ-producing cells was calculated.
Data are representative of three experiments.
3.16 The butanol fraction of B. pilosa prevents the onset of diabetes in NOD mice Th1 cells were reported to cause insulitis and diabetes in NOD mice (Katz et al., 1995). Our in vitro data exhibited that the butanol fraction of B. pilosa could suppress the differentiation of Th0 into Th1 cells and preferentially promote that of Th0 into Th2 cells, implying a potential role of the butanol fraction in treating Th1-mediated autoimmune diseases. We reasoned that the butanol fraction may in vivo prevent diabetes in NOD mice via down-regulation of Th1 cells or up-regulation of Th2 cells, which antagonize Th1 cell function. To test this hypothesis, we utilized NOD mice as a Th1-mediated autoimmune disease mouse model to examine the effect of the butanol fraction in the diabetes progression. Our results indicated that mice with intraperitoneal (i.p.) injection using the butanol fraction at 3 mg/kg per dose had a lower diabetes incidence (33%) than control mice (56%), which has a similar incidence in the publication (Kai et al., 1993). Likewise, injection of NOD mice with the butanol fraction at 10 mg/kg could stop the initiation of the disease (0%). Thus, the butanol fraction treatment could protect NOD mice from developing diabetes in a dose-dependent manner (Figure 24). We also examined diabetes indicators such as blood glucose and insulin. We found that the butanol fraction treatment at 10 mg/kg prevented mice from hyperglycemia and hypoinsulinemia in comparison to control mice (Figure 25).
Figure 24. The effect of butanol fraction on the diabetes progression in NOD mice. Cumulative diabetes incidence in female NOD mice. Three groups of female NOD mice (mouse numbers per group are indicated in the parenthesis) received i.p.
injections with the butanol fraction of B. pilosa at 3 mg/kg (BuOH 3 mg/kg), 10 mg/kg (BuOH 10 mg/kg) or PBS (Control) 3 times per week from 4 to 27 weeks of age.
Urine glucose was monitored using Clinistix at the indicated ages. Mice with 28 mM glucose or more in their urine for two consecutive weeks were considered to be diabetic.
Figure 25. The effect of butanol fraction on blood glucose and blood insulin level in NOD mice. (A) The concentration of blood glucose (mg/dl) of the 10 mg/kg butanol fraction of B. pilosa-treated (BuOH 10 mg/kg) or control mice (4, 15 and 18 weeks of age) was determined using a glucometer. Data (mean ± s.e.) are representative of three experiments. (B) The same mice as indicated in panels A had their blood insulin concentrations (pg/ml) determined using an ELISA kit. Data (mean
± s.e.) are representative of three experiments and * : P < 0.05 by Student T test..
3.17 Two polyacetylenic compounds, identified from butanol fraction of B. pilosa, protect the diabetes onset in NOD mice.
We next determined if the bioactive phytocompounds identified from the the butanol fraction have similar effects on prevention of diabetes as the butanol fraction.
We found that treatment with these two compounds significantly prevented the onset of diabetes in NOD mice (Table I).
Table I. Effect of the polyacetylenic compounds on diabetes prevention.
Compound 1 (n=3) 0% diabetic Compound 2 (n=5) 0% diabetic Control (n=6) 33% diabetic
NOD mice were i.p. injected 3 times per week from 10 to 13 weeks of age with different amounts of compound 1 (37 μg/kg) or compound 2 (45 μg/kg). The definition of diabetes is described in the legend of Figure 24. n : mouse number.
Chapter 4. Discussion
4.1 Bioactives from B. pilosa
Natural products have been the most productive source of leads for the drug development. More than 80% of drug substances were natural products or inspired by a natural compound. It’s about 50% of the drugs approved from 1994 to 2007 are based on natural products.
B. pilosa has been claimed as an anti-infectious or immunomodulatory folk
medicine. Here, we first evaluated the immune efficacy of B. pilosa, as evidenced in the up-regulation of IFN-γ, a potent cytokine in many immunomodulatory aspects. We have effectively identified two bioactive flavonoids, centaurein and centaureidin, from B. pilosa with the ability to stimulate IFN-γ expression using a BGFI method. It was
reported that centaurein and centaureidin were synthesized in B. pilosa or other plants (Chiang et al., 2004). However, their biological functions remained unknown. In this study, we, for the first time, manifested that centaurein and its aglycone, centaureidin, were able to modulate IFN-γ transcription. Centaurein was used to further study how both flavonoids could stimulate IFN-γ transcription. Our results showed that centaurein could activate the transcription activity of T-bet. Therefore, we postulated that centaurein mediated IFN-γ expression through nuclear factor T-bet. Our data manifested that centaurein or centaureidin can in vitro boost IFN-γ production. Their
in vivo function in immune modulation (e.g., pathogen clearance) was further verified
using Listeria infection mouse models.
We here proved the concept that a combination of IFN-γ promoter, luciferase as a reporter gene, and T cells can be used to screen immunomodulatory phytochemicals from the B. pilosa plant, traditionally used as a folk medicine to improve immunity and infections. Although the use of luciferase as a reporter gene in biological assays is not a brand-new idea, yet it is relatively rapid, sensitive, cost-effective, and feasible for robotization (New et al., 2003). The effective dose of centaurein used here to stimulate the IFN-γ production is relative high (100 μg/ml). In contrast, centaureidin, an aglycone of centaurein, at 2 μg/ml has similar effect as centaurein at 100 μg/ml on IFN-γ stimulation. Of note, we proved that compounds with a low or high potency were able to be identified in our experimental setups.
On the other hands, our results showed that two polyacetylenic compounds and a butanol fraction of B. pilosa extract modulated T cell differentiation. Meanwhile, the butanol fraction also lowered diabetic incidence, whilst maintaining normal levels of blood sugar, insulin produced by β cells in NOD mice. To our knowledge, this is the first report so far to demonstrate that the butanol fraction of B. pilosa can effectively prevent IDDM, as evaluated using a NOD mouse model. One possible scenario for the suppression of IDDM may be that the butanol fraction and polyacetylenic
compounds inhibit the generation of Th1 cells and promotes that of Th2 cells infiltrating into the islets of NOD mice as the fraction and compounds do in vitro.
Intriguingly, B. pilosa has been used as herbal medicines to treat diabetes without scientific proof (Dimayuga and Agundez, 1986). A mixture of 2-β-D-glucopyranosyloxy-1-hydroxy-5(E)-tridecene-7,9,11-triyne (1) and 3-β-D-glucopyranosyloxy-1-hydroxy-6(E)-tetradecene-8,10,12-triyne (2) from B.
pilosa have been demonstrated to have blood sugar lowering effect in type II diabetes
(db/db) mice, partly ascribed to the anorexic effect of both polyacteylenic glucosides (Ubillas et al., 2000). However, the mechanism by which both polyacetylenic glucosides affect diabetes in the type II diabetes mice is not elucidated.
4.2 Listeria infection
The annual incidence of listeriosis in humans is rare, ~ 0.4 to 7.4 per million people (Calder, 1997). But among those who develop listeriosis, the death rate is high (~25 to 30%) (Rouquette and Berche, 1996). Antibiotic drugs are currently used for
Listeria infection. However, antibiotic-resistant Listeria strains have been increasingly reported (Yamaoka et al., 1998; Yamaoka et al., 2000). Therefore, it is necessary to develop new therapeutics for treating Listeria infection.
Listeria infection in mice. For example, the crude extract of a Chinese medicine,
Bu-Zhong-Yi-Qi-Tang, up-regulates IFN-γ production and, therefore, eradicates
Listeria infection in mice (Yamaoka et al., 1998; Yamaoka et al., 2000; Yamaoka et al.,
2001). Some polysaccharides isolated from the Echinacea purpurea plant protect mice against Listeria infection (Steinmuller et al., 1993). However, the detailed pharmacological mechanisms and/or the bioactive compounds in the above systems remain to be elucidated.
Centaurein was previously isolated from a plant (B. pilosa) with a folk tradition of anti-bacterial use (Chiang et al., 2004). Here, we, for the first time, found that NK and T cells increase IFN-γ production in response to centaurein. This IFN-γ increase was also observed in mice. The fact that centaurein up-regulates T-bet expression suggests a molecular mechanism by which centaurein mediates IFN-γ expression via an IFN-γ regulator, T-bet. In this study, we confirm that centaurein can protect against or treat Listeria infection in mice via up-regulation of IFN-γ and macrophage activation.
IFN-γ can activate the macrophage-mediated killing of intracellular pathogens.
Both IFN-γ and macrophage activation are pivotal for Listeria eradication in cell and animal models (Hubel et al., 2002). IFN-γ alone or in conjunction with antimicrobial agents, is also reported to clinically treat patients infected with an intracellular
microbe, Mycobacteria (Hubel et al., 2002). Of note, IFN-γ showed a promising effect on the adjunctive treatment of multidrug-resistant Mycobacteria in patients (Hubel et al., 2002). We report that centaurein alone or in combination with antibiotics protects against and treats Listeria infection via up-regulation of IFN-γ. In addition, the anti-bacterial susceptibility test showed that the minimal inhibitory concentration of centaurein for Listeria is over 200 μg/ml, indicating that centaurein itself did not show any significant bacteriocidal or bacteriostatic activity against Listeria because of its high minimal inhibitory concentration. On the contrary, centaurein can prevent and treat Listeria infection indirectly via boosting immune responses (IFN-γ production and macrophage activation).
Our results are encouraging for the use of centaurein protecting against and treating antibiotic-resistant intracellular bacteria via enhanced IFN-γ production.
Similar approaches can be used to develop immune modulators and prophylactics/therapeutics for infectious pathogens.
4.3 Autoimmune diseases
Autoimmune disease is the third largest category of illness in the developed countries—behind cardiovascular disease and cancer. It was estimated that over 20 million people are afflicted with autoimmune diseases and that a conservative medical expenditure covering those autoimmune diseases is 21 billion US dollars per year.
However, few drugs have so far been developed for autoimmune diseases compared to other diseases. Treatments for autoimmune diseases rely on immunosuppressants or immune modulators. Immunosuppressants such as cyclosporine A can shut down the immune system and prevent inflammation. However, treatment with immunosuppressants may carry a risk of infectious disease or cancers (Kai et al., 1993). Immune modulators, which skew T cell differentiation, have been used to treat T cell-mediated disorders. For instance, IL-4 and IL-10 are used for treatment of Th1- mediated diseases (Kawamoto et al., 2001; Ko et al., 2001). However, a strategy which skews production of Th1 cells into Th2 cells may have adverse effects, such as the induction of Th2-mediated autoimmune diseases.
Our study is designed to identify immunomodulatory plant extracts and phytocompounds using in vitro T cell differentiation method in combination with NOD mouse model. Here, we have successfully screened out a butanol fraction of B.
pilosa and its subsequent two compounds which can suppress Th1 differentiation but
promote Th2 differentiation from Th0 cells. Our results from NOD mouse model also demonstrated that the butanol fraction and compounds can prevent the progress of Th1-mediated diabetes. Our results are encouraging the potentially therapeutic use of butanol fraction and bioactive compounds in Th1-mediated autoimmune diseases.
Chapter 5. Conclusions and future perspectives
Our results demonstrated that both T cell-based luciferase reporter assay and T helper differentiation assay can be used to identify immunomodulatory phytochemicals from the B. pilosa. These screening methods may be further improved and developed into a high throughput platform for evaluating and screening other immunomodulatory herbs, fractions and compounds.
We demonstrate that centaurein protects against or treats Listeria infection through a regulation of IFN-γ expression. However, in our studies, the beneficial therapeutic effect of centaurein was only based on healthy young mice and/or IFN-γ knockout mice with serious innate immunodeficiency. Additional experiments in evaluating the efficacy of centaurein in partially immunocompromised mice, such as dexamethasone-treated mice, needs be considered. The use of healthy mice and mice with innate or acquired immunodeficiency to evaluate the therapeutic effect of centaurein on Listeria infection help draw the conclusion on the efficacy of centaurein in Listeria elimination and may be more like humans susceptible to Listeria infection.
Our data showed that centaurein increased the IFN-γ production in T and NK cells. T-bet is required for IFN-γ production in T cells and NK cells (Szabo et al., 2002; Townsend et al., 2004). However, T-bet was reported not to be required for host resistance to Listeria infection (Way and Wilson, 2004). Our results showed that
centaurein augments IFN-γ expression in cells and mice. Such an increase accompanies T-bet up-regulation. Therefore, our results strongly suggest that centaurein elevates IFN-γ production via control of T-bet. IFN-γ was reported to induce T-bet expression (Lighvani et al., 2001), raising the possibility that the up-regulation of T-bet is an indirect consequence of centaurein inducing IFN-γ.
However, this should not be the case because centaurein still up-regulates T-bet transcription in Jurkat cells, in which IFN-γ production is defective. The regulation of IFN-γ expression involves a complicated mechanism mediated by various nuclear factors. More studies utilizing the T-bet knock out mice or primary cells from T-bet knock out strain would provide additional evidences to ascertain the detail mechanism of the action of centaurein on Listeria infection and position the involvement of T-bet in the production of IFN-γ in response to centaurein.
In additional, PHA, a T cell stimulant, can activate T cells to produce cytokines like IFN-γ and then cause T cell death. This phenomenon is known as activation-induced cell death (Chwae et al., 2002). We also observed that similar to PHA, centaurein and centaureidin in some cases induced IFN-γ transcription as well as apoptosis in T cells. However, PHA and both flavonoids at the same dose showed a marginal effect on the cell death of a non-T cell line, COS cells, suggesting that the significant effect of the above compounds on T cell death was partially ascribed to
activation-induced cell death. How both flavonoids can cause T cell activation-induced death needs to be further examined.
The concentrations of centaurein used in our studies are 100 μg/ml in cells and 10 to 20 μg/mouse in mice. Additionally, our data showed that centaureidin, an aglycone of centaurein, could increase IFN-γ production 30 times more than centaurein. Therefore, there is great potential for use of centaurein or its derivatives to treat infectious diseases.
Identification of bioactive pure compounds from B. pilosa can help us elucidate the mechanism by which B. pilosa can prevent non-obese diabetes. Here, we demonstrated that the butanol fraction of B. pilosa can ameliorate type I diabetes probably via controlling T cell differentiation into Th2 cells. Indeed, we have identified two pure compounds (compound 1 and compound 2) which can prevent diabetes development in NOD mice although compound 2 is more potent than compound 1 in T cell differentiation. More studies are warranted to ascertain the detailed mechanism of the action of these compounds on IDDM.
B. pilosa was documented to treat other categories of diseases. For instance, B.
pilosa extracts was shown to decrease acid/pepsin secretion (Alvarez et al., 1999) and inhibit ulcers (Tan et al., 2000). Besides, its extract was shown to have anti-hypertensive effects in rats (Dimo et al., 2001; Dimo et al., 1999), inhibit the
vasocontriction by blocking the Ca2+ influx into the cells (Dimo et al., 1998;
Nguelefack et al., 2005) and slow cardiac pump (Dimo et al., 2003). However, no specific compound responsible for the above categories of diseases of B. pilosa has been identified up to date. Bioactivity-directed fractionation and isolation approach can be taken to understand the active compounds of B. pilosa for gastrointestinal and cardiovascular diseases.
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