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Baicalin induced dendritic cell apoptosis in vitro.

Zhang H, Jiao Q, Gong Q, Zhang Y, Zhang W, Hu Z - Front Pharmacol (2011)

Bottom Line: This study was aimed to investigate the effects of baicalin (BA), a major flavonoid constituent found in the herb Baikal skullcap, on dendritic cells (DCs).Exposure of DCs to BA (2-50 μM) during BM cell differentiation showed no effects on the up-regulation of CD80/CD86 expression on DCs in response to LPS stimulation, but reduced DCs recovery by inducing apoptosis, and significantly inhibited the release of IL-12 to culture supernatants.These results demonstrate that BA induces selective apoptosis in immature DCs possibly through mitochondria-mediated pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, Second Military Medical University Shanghai, China.

ABSTRACT
This study was aimed to investigate the effects of baicalin (BA), a major flavonoid constituent found in the herb Baikal skullcap, on dendritic cells (DCs). DCs were generated by culturing murine bone marrow (BM) cells for 6 days with granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4, and lipopolysaccharide (LPS) was added on day 5 to stimulate DCs maturation. The expression levels of DC maturity markers (CD80/CD86) were assessed by flow cytometry using direct immunofluorescence method. IL-12 levels in the culture supernatants were assayed by ELISA. Apoptosis of DCs was analyzed by flow cytometry after annexin V/propidium iodide staining. The mitochondrial membrane potential (Δψ(m)) changes were measured by using the J-aggregate forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Exposure of DCs to BA (2-50 μM) during BM cell differentiation showed no effects on the up-regulation of CD80/CD86 expression on DCs in response to LPS stimulation, but reduced DCs recovery by inducing apoptosis, and significantly inhibited the release of IL-12 to culture supernatants. BA-induced DC apoptosis in a time- and dose-dependent way, and immature DCs were more sensitive for BA-induced apoptosis than mature DC. BA also induced Δψ(m) changes in DCs. These results demonstrate that BA induces selective apoptosis in immature DCs possibly through mitochondria-mediated pathway.

No MeSH data available.


Related in: MedlinePlus

Effects of BA on bone marrow-derived DC (BMDC) maturation and apoptosis. (A) Effects of BA on the up-regulation of CD80/CD86 expressions on day-6 BMDCs in response to LPS stimulation. (B) Effect of BA on numbers of viable CD11c+ cells recovered on day 6 of culture BM cells (expressed as percentages of total viable number of CD11c+ cells in non-treatment control). (C) Effect of BA on the release of IL-12 into supernatants. (A–C) Results were obtained from three independent experiments and presented as mean values (±SD). *p < 0.05 vs. LPS alone control by Student's t-test. (D) Representative histograms showing CD80 and CD86 expression on day-6 BMDCs from three independent experiments. (E) The percentage of apoptosis in day-6 BMDCs determined by FACS, using annexin V/PI staining. The results are representative of three independent experiments.
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Figure 2: Effects of BA on bone marrow-derived DC (BMDC) maturation and apoptosis. (A) Effects of BA on the up-regulation of CD80/CD86 expressions on day-6 BMDCs in response to LPS stimulation. (B) Effect of BA on numbers of viable CD11c+ cells recovered on day 6 of culture BM cells (expressed as percentages of total viable number of CD11c+ cells in non-treatment control). (C) Effect of BA on the release of IL-12 into supernatants. (A–C) Results were obtained from three independent experiments and presented as mean values (±SD). *p < 0.05 vs. LPS alone control by Student's t-test. (D) Representative histograms showing CD80 and CD86 expression on day-6 BMDCs from three independent experiments. (E) The percentage of apoptosis in day-6 BMDCs determined by FACS, using annexin V/PI staining. The results are representative of three independent experiments.

Mentions: The effect of BA on DCs was initially investigated in BM cell cultured for 6 days with GM-CSF and IL-4, which generates mainly BMDCs. BA (0–50 μM) was added on day 3, and LPS (500 ng/ml) was added on day 5 to stimulate DC maturation. The surface expressions of CD11c, CD80, and CD86 on BA-treated and -untreated DCs were examined by flow cytometry, and levels of IL-12 in the supernatants were measured by ELISA. The results demonstrated that more than 90% of non-adherent and loosely adherent cells were CD11c+ cells, and LPS stimulation markedly up-regulated the expression of CD80 and CD86 within the CD11c+ population (Figures 2A,D) and induced substantial release of IL-12 to the culture supernatants (Figure 2C), suggesting most cells harvested on day 6 were DCs. BA inclusion showed no effects on the up-regulation of CD80/CD86 expression in response to the stimulation of LPS (Figures 2A,D), but resulted in a concentration-dependent reduction of total numbers of viable CD11c+ cells recovered on day 6 of culture (Figure 2B), and significantly suppressed the release of IL-12 to culture supernatants (Figure 2C). To examine whether BA reduced cell recovery by inducing cell death, apoptosis in the day-6 DCs was further assessed by FACS after annexin V/PI staining. A significant degree of apoptosis was detected in the BA-treated DC, as shown by an increased phosphatidyl exposure using annexin V-FITC (Figure 2E).


Baicalin induced dendritic cell apoptosis in vitro.

Zhang H, Jiao Q, Gong Q, Zhang Y, Zhang W, Hu Z - Front Pharmacol (2011)

Effects of BA on bone marrow-derived DC (BMDC) maturation and apoptosis. (A) Effects of BA on the up-regulation of CD80/CD86 expressions on day-6 BMDCs in response to LPS stimulation. (B) Effect of BA on numbers of viable CD11c+ cells recovered on day 6 of culture BM cells (expressed as percentages of total viable number of CD11c+ cells in non-treatment control). (C) Effect of BA on the release of IL-12 into supernatants. (A–C) Results were obtained from three independent experiments and presented as mean values (±SD). *p < 0.05 vs. LPS alone control by Student's t-test. (D) Representative histograms showing CD80 and CD86 expression on day-6 BMDCs from three independent experiments. (E) The percentage of apoptosis in day-6 BMDCs determined by FACS, using annexin V/PI staining. The results are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3108619&req=5

Figure 2: Effects of BA on bone marrow-derived DC (BMDC) maturation and apoptosis. (A) Effects of BA on the up-regulation of CD80/CD86 expressions on day-6 BMDCs in response to LPS stimulation. (B) Effect of BA on numbers of viable CD11c+ cells recovered on day 6 of culture BM cells (expressed as percentages of total viable number of CD11c+ cells in non-treatment control). (C) Effect of BA on the release of IL-12 into supernatants. (A–C) Results were obtained from three independent experiments and presented as mean values (±SD). *p < 0.05 vs. LPS alone control by Student's t-test. (D) Representative histograms showing CD80 and CD86 expression on day-6 BMDCs from three independent experiments. (E) The percentage of apoptosis in day-6 BMDCs determined by FACS, using annexin V/PI staining. The results are representative of three independent experiments.
Mentions: The effect of BA on DCs was initially investigated in BM cell cultured for 6 days with GM-CSF and IL-4, which generates mainly BMDCs. BA (0–50 μM) was added on day 3, and LPS (500 ng/ml) was added on day 5 to stimulate DC maturation. The surface expressions of CD11c, CD80, and CD86 on BA-treated and -untreated DCs were examined by flow cytometry, and levels of IL-12 in the supernatants were measured by ELISA. The results demonstrated that more than 90% of non-adherent and loosely adherent cells were CD11c+ cells, and LPS stimulation markedly up-regulated the expression of CD80 and CD86 within the CD11c+ population (Figures 2A,D) and induced substantial release of IL-12 to the culture supernatants (Figure 2C), suggesting most cells harvested on day 6 were DCs. BA inclusion showed no effects on the up-regulation of CD80/CD86 expression in response to the stimulation of LPS (Figures 2A,D), but resulted in a concentration-dependent reduction of total numbers of viable CD11c+ cells recovered on day 6 of culture (Figure 2B), and significantly suppressed the release of IL-12 to culture supernatants (Figure 2C). To examine whether BA reduced cell recovery by inducing cell death, apoptosis in the day-6 DCs was further assessed by FACS after annexin V/PI staining. A significant degree of apoptosis was detected in the BA-treated DC, as shown by an increased phosphatidyl exposure using annexin V-FITC (Figure 2E).

Bottom Line: This study was aimed to investigate the effects of baicalin (BA), a major flavonoid constituent found in the herb Baikal skullcap, on dendritic cells (DCs).Exposure of DCs to BA (2-50 μM) during BM cell differentiation showed no effects on the up-regulation of CD80/CD86 expression on DCs in response to LPS stimulation, but reduced DCs recovery by inducing apoptosis, and significantly inhibited the release of IL-12 to culture supernatants.These results demonstrate that BA induces selective apoptosis in immature DCs possibly through mitochondria-mediated pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, Second Military Medical University Shanghai, China.

ABSTRACT
This study was aimed to investigate the effects of baicalin (BA), a major flavonoid constituent found in the herb Baikal skullcap, on dendritic cells (DCs). DCs were generated by culturing murine bone marrow (BM) cells for 6 days with granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4, and lipopolysaccharide (LPS) was added on day 5 to stimulate DCs maturation. The expression levels of DC maturity markers (CD80/CD86) were assessed by flow cytometry using direct immunofluorescence method. IL-12 levels in the culture supernatants were assayed by ELISA. Apoptosis of DCs was analyzed by flow cytometry after annexin V/propidium iodide staining. The mitochondrial membrane potential (Δψ(m)) changes were measured by using the J-aggregate forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Exposure of DCs to BA (2-50 μM) during BM cell differentiation showed no effects on the up-regulation of CD80/CD86 expression on DCs in response to LPS stimulation, but reduced DCs recovery by inducing apoptosis, and significantly inhibited the release of IL-12 to culture supernatants. BA-induced DC apoptosis in a time- and dose-dependent way, and immature DCs were more sensitive for BA-induced apoptosis than mature DC. BA also induced Δψ(m) changes in DCs. These results demonstrate that BA induces selective apoptosis in immature DCs possibly through mitochondria-mediated pathway.

No MeSH data available.


Related in: MedlinePlus