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PARACTIN® inhibits IFNg and IL-2 cytokines production
and protects against cell apoptosis
1Juan L. Hancke, & 1Rafael A. Burgos, Karina Seguel ,Mirna Perez,
Ada Meneses, Marcele Ortega
Several clinical trials performed with PARACTIN® have demonstrated that this plant extract possessed efficacy for the treatment of uncomplicated respiratory tract infections. The main active compounds, andrographolides, have demonstrated anti-inflammatory properties such as inhibition of nitrite synthesis by suppressing of iNOS expression in RAW 264.7 cells, prevention of reactive oxygen species (ROS) production and reduction of adhesion molecules in neutrophils. All these studies indicated that andrographolide can reduce the activity of macrophages and neutrophils, which is related to innate immune response. The anti-inflammatory effect of andrographolide on the adaptive immune response is unknown, despite the fact that other labdane diterpenic compounds have shown immunosuppressant activity. In this study we present evidence that andrographolide in PARACTIN® reduces the IFN? and IL-2 production in murine T-cells and protects against cell apoptosis.
The T-cells and thymocytes were isolated from 6-to8-week old RK mice. The. Lymphocytes were obtained from lymph nodes. T-cells were incubated with concanavaline A (CON-A) and various concentration of andrographolide or 14 DAP. The supernatant was collected and the IFN?, IL-2 and IL-4 production was measured using ELISA. The ERK1/2 phosphorylation was analyzed and apoptosis was quantified by DNA fragmentations.
IFN-? and IL-2 release T-cells was induced after 24h of incubation with 3.3 µg/mL of CON-A. All doses of androgapholide inhibited dramatically the CON-A rise of IFN-? with maximum inhibitory response at doses of 7.5µM and IC50 1.7±0.07µM. 14-DAP was also able to inhibit the IFN- ? production in CON-A stimulated cells, but not as strongly as andrographolide. Doses of 15 µM of 14-DAP showed the maximum inhibitory response in CON-A stimulated T-cells and IC50 of 35.8 ±0.50 µM. On the other hand, andrographolide but not 14-DAP inhibited the IL-2 production induced by CON-A. The IL-4 production was not modified wither by andrographolide or 14-DAP in T-cells stimulated with CON-A with the same concentrations that reduced IFN? (data not shown).
Since the inhibitory effect of andrographolide was more evident on IFN-?, we developed an RT-PCR that showed a strong inhibition of mRNA for IFN-? in T-cells stimulated with 3.3µg/mL of CON-A at lower concentrations 0.01-0.5µM and 5nM inhibited completely the increase of mRNA (data not shown).
The expression ICAM-1, iNOS, COX-2 and proinflammatory cytokine are controlled by mitogen-activated proteinkinase (MAPK). Our results show that andrographolide in PARACTIN® (5 and 10µM) reduced the ERK1/2 phosphorylation induced by CON-A. ERK 1/2 is a MAPK that requires phosphorylation to become active. 14-DAP in PARACTIN® inhibited ERK1 phosphorylation; however, ERK2 phosphorylation was only partially inhibited.
NF-kB has been described to regulate the cellular process of apoptosis. Apoptosis is a mechanism that could explain the reduction of the immune response. The DNA fragmentation of thymocytes was incubated with hydrocortisone and PMA. Andrographolide and 14-DAP in PARACTIN® alone did not induce an increase in apoptosis. 50µM of andrographolide from PARACTIN® reduced the apoptosis induced by hydrocortisone up to 20%. On the other hand, inhibition of thymocytes apoptosis with 14-DAP was only observed at doses of 100µM. 100µM of andrographolide from PARACTIN® significantly reduced the apoptosis induced by PMA up to 35%. 14-DAP, at all tested concentrations, and had no effect on PMA-induced apoptosis. Apoptosis induced by hydrocortisone increases the production of caspase-3 activity. Andrographolide from PARACTIN® at doses of 50 and 100µM was able to reduce significantly the spontaneous caspase-3 like activity.
We conclude that andrographolide and to a lesser extent 14-DAP from PARACTIN® extract, inhibit significantly the production of IFN? in T-ells and ERK1/2 phosphyloration and protects against apoptosis in thymocytes. The immunosuppressant effect of andrographolide in PARACTIN® opens a potential use in several autoimmune diseases associated with an increase in IFN? such as multiple sclerosis, rheumatoid arthritis, psoriasis and autoimmune diabetes.
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