Schizandra arisanensis extract attenuates cytokine-mediated cytotoxicity in insulin-secreting cells

Figure 1 Chemical fingerprinting of the ethanolic extract of Schizandra arisanensis. The ethanolic extract of Schizandra arisanensis (SA-Et) (1.0 mg) was subjected to a high-performance liquid chromatography system to obtain chemical fingerprints. According to the retention times, the chemical identities and structures of seven major peaks are listed.

Figure 2 Interleukin-1β and interferon-γ-mediated BRIN-BD11 cell apoptosis is attenuated by p38MAPK and stress-activated protein kinase/c-Jun NH2-terminal kinase inhibitors. A: The viability of interferon (IFN)-γ (100 ng/mL)-treated cells in the presence of various concentrations of interleukin (IL)-1β for 24 h, 48 h and 72 h was measured. Data are presented as the mean ± SE, n = 4. ^aP < 0.05, ^bP < 0.01 vs the viability of untreated cells; B: Cell cycle analysis was carried out at 48 h post-treatment with IL-1β (5 ng/mL), IFN-γ (100 ng/mL), or a mixture of the two. Data are presented as the mean ± SE, n = 4. ^aP < 0.05, ^bP < 0.01 vs the control; C: The viability of cytokine-treated BRIN-BD11 cells in the presence of inhibitors was measured. Data are presented as the mean ± SE, n = 4. ^bP < 0.01 vs the viability of untreated cells; ^dP < 0.01 vs the viability of cells with the cytokine mix.

Figure 3 The anti-apoptotic effect of the ethanolic extract of Schizandra arisanensis. A: The viability of BRIN BD11 cells was determined after 48 h of cytokine treatment in the presence or absence of the ethanolic extract of Schizandra arisanensis (SA-Et); B: Cell cycle of BRIN BD11 cells was determined after 48 h of cytokine treatment in the presence or absence of the SA-Et. Data are presented as the mean ± SE, n = 4. ^aP < 0.05, ^bP < 0.01 vs the control; C: Both full and cleaved forms of caspase-3 protein were analyzed by Western blot after 24 h of cytokine treatment in the presence or absence of the SA-Et. A representative from three experiments is shown. IL-1β: Interleukin 1β; IFN-γ: Interferon-γ.

Figure 4 Characterizing the impact of ethanolic extract of Schizandra arisanensis on cytokine signal transduction. A: BRIN-BD11 cells stimulated with the cytokine mix in the presence or absence of ethanolic extract of Schizandra arisanensis (SA-Et) (20 μg/mL) were harvested after 30 min treatment for Western blot with the indicated antibodies. A representative Western blot from three experiments is shown; B: BRIN-BD11 cells stimulated with the cytokine mix in the presence or absence of SA-Et were harvested after 60 min treatment to measure IκBα protein level. A representative Western blot from three experiments is shown; C: Cytokine-induced inducible nitric oxide synthase (iNOS) mRNA levels in BRIN-BD11 cells during 24 h were determined by an reverse transcription-polymerase chain reaction. Relative iNOS gene expression was calculated by employing endogenous β-actin mRNA level as an internal control. The maximum iNOS gene expression at 4 h post-treatment with the cytokines alone was set to 100%. Data are presented as the mean ± SE, n = 4; D: Changes in cytokine-induced iNOS mRNA levels in BRIN-BD11 cells at 4 h in the presence or absence of the SA-Et (20 μg/mL) were also determined by real-time reverse transcription-polymerase chain reaction. β-actin was used as an internal control. The relative quantification of iNOS mRNA was presented as 2^-ΔΔct. Data are presented as the mean ± SE, n = 3. ^bP < 0.01 vs cells under control conditions; E: Nitric oxide production in cytokine-treated BRIN-BD11 cells in the presence or absence of the SA-Et (0-20 μg/mL) or nitro-L-arginine methyl ester (L-NAME) (0.5 mmol/L) was determined. Data are presented as the mean ± SE, n = 4. ^bP < 0.01 vs cells treated with the cytokine mixture alone. IL-1β: Interleukin 1β; IFN-γ: Interferon-γ.

Figure 5 Ethanolic extract of Schizandra arisanensis treatment partially rescued the abolished insulin secretion in cytokine-treated BRIN-BD11 cells. A: Acute insulin release in response to glucose, KCl and Ca²⁺ in the presence or absence of the ethanolic extract of Schizandra arisanensis (SA-Et) was evaluated. Data are presented as the mean ± SE, n = 4. ^aP < 0.05, ^bP < 0.01 vs insulin released from cells treated with 1.1 mmol/L glucose; ^dP < 0.01 vs control cells under the same conditions; B: Cultured BRIN-BD11 cells were treated with the cytokine mixture in the presence or absence of the SA-Et. At 48 h post-treatment, treated cells were evaluated for glucose responsiveness. Data are presented as the mean ± SE, n = 4. ^bP < 0.01 vs insulin released from cells treated with 1.1 mmol/L glucose; ^dP < 0.01 vs SA-Et-treated cells under the same glucose conditions. C: In addition, a portion of the treated cells was harvested to measure insulin mRNA level using reverse transcription-polymerase chain reaction. Relative insulin gene expression was calculated by employing endogenous β-actin mRNA level as an internal control. ^aP < 0.05 vs control conditions (none); D: Protein level and cellular insulin content were independently measured by Western blot and enzyme-linked immunosorbent assay. Data are presented as the mean ± SE, n = 4. ^aP < 0.05, ^bP < 0.01 vs control conditions (none). IL-1β: Interleukin 1β; IFN-γ: Interferon-γ; ND: Not determined.

Figure 6 Identification of bio-active compounds isolated from Schizandra arisanensis. After major peak compounds were collected, BRIN-BD11 cells were treated with cytokine mix in the presence of each peak compounds for 48 h. The viability of cells was then measured by neutral red assay. Epigallocatechin gallate (EGCG) is used as reference drug. Data are presented as the mean ± SE, n = 8. ^bP < 0.01 vs the viability of cells with no treatment.