Fig 1.
Chemical structures of Schisandrin A and Schisandrin B.
Fig 2.
Hypotheses of the present study.
Fig 3.
Experimental design of the present study.
Fig 4.
Effect of Sch A and Sch B incubation on glutathione S-transferase (GST) activity and reduced glutathione (GSH) levels in RAW264.7 macrophages.
RAW264.7 macrophages were exposed to Sch A and Sch B [alone (25 and 50 μM) or in combination (25 μM)], as described in Materials and Methods. (A) After 1 h incubation, cells were harvested and lysed for the measurement of GST activity. (B) The cellular GSH level was measured at 6 hour post-incubation. Data expressed as % control, by normalizing relative to the value of control (vehicle). Value given are means ± SEM, with n = 6. * Significantly different from the control.
Fig 5.
Effect of a 6-h incubation with Sch A and Sch B in lipopolysaccharide (LPS)-activated pro-inflammatory signals in RAW264.7 macrophages.
After incubation with Sch A and Sch B for 6 h, RAW264.7 macrophages were challenged with LPS (1 μg/mL) for 15 min. (A, B) The levels of phospho-JNK1/2 (and phospho-p38) and total-JNK1/2 (and total p38) were measured using Western blot analysis. The ratio of phoshos-JNK1/2 to total-JNK1/2 as well as the ratio of phospho-p38 to total p38 (which are indicative of JNK1/2 and p38 activation) were estimated and expressed as % control, by normalizing with the value of the non-LPS group (JNK1/2: left panel, p38: right; upper panel). (C) After transfection with NF-κB-luciferase reporter in RAW264.7 macrophages, the RAW264.7 cells were incubated with Sch A or Sch B, as described in Materials and Methods. NF-κB-luciferase reporter, which consists of an NF-κB- responsive firefly luciferase construct and a constitutively expressed Renilla luciferase. Following exposure to LPS for 1 h, luciferase activities in the cell lysates were measured. Renilla luciferase expression was used to normalize the varying transfection efficiencies among the samples. NF-κB reporter activity was expressed as % control, by normalizing with the value of the non-LPS control. Value given are means ± SEM, with n = 3. * Significantly different from the non-LPS control; # significantly different from the LPS control.
Fig 6.
Effect of a 6-h incubation with Sch A and Sch B in lipopolysaccharide (LPS)-activated pro-inflammatory effectors in RAW264.7 macrophages.
(A, B) Following incubation with Sch A or Sch B, RAW264.7 macrophages were exposed to LPS for 2 h. RNA of the cell lysate was isolated using a RNA isolation kit. The isolated RNA was converted into cDNA using a cDNA synthesis kit. The cDNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) were measured by real time polymerase chain reaction using TaqMan Array plates on the 7500 Fast system. The value of cycle threshold (CT value) was calculated. The CT values (which are indicative of relative cDNA levels) of iNOS and COX2 were normalized with the respective CT value of internal control (18S ribosomal RNA). Data are expressed as % non-LPS control by normalizing relataive to the value of non-LPS stimulated cells (Upper panel). (C, D) Following exposure to LPS for 18 h, the levels of iNOS and COX2 were measured using Western blot analysis. The levels of iNOS and COX2 were normalized relative to β-actin and expressed as % non-LPS control (middle panel). (E, F) Following exposure to LPS for 24 h, the levels of nitric oxide (NO, the product of an iNOS-catalyzed reaction) and prostaglandin E2 (PGE2, the product of a COX2-catalyzed reaction) were measured by Griess assay and ELISA, respectively. Data are expressed as % non-LPS control by normalizing relative to non-LPS stimulated cells (lower panel). Values given are means ± SEM, with n = 3–5. * Significantly different from the non-LPS control; # significantly different from the LPS control.
Table 1.
Effect of 6-h incubation with Sch A and Sch B in lipopolysaccharide (LPS)-activated pro-inflammatory cytokines in RAW264.7 macrophages.
Table 2.
Effects of GST inhibitor and thiol/non-thiol antioxidants on the anti-inflammatory actions produced by Sch A and Sch B in lipopolysaccharide-activated RAW264.7 macrophages.
Fig 7.
Effect of Sch A and Sch B incubation on the antioxidant response in RAW264.7 macrophages.
RAW264.7 macrophages were incubated with Sch A or Sch B for 6 h. (A) Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master transcription factor for the antioxidant system. Levels of Nrf2 and β-actin in cytosolic fractions as well as Nrf2 and lamin B1 in nuclear fractions of macrophages were measured using Western blot analysis. Whereas the level of Nrf2cytosolic was normalized relative to the β-actincytosolic level, Nrf2nuclear content was normalized relative to the lamin-B1nuclear level. The ratio of normalized nuclear Nrf2 to normalized cytosolic Nrf2 was determined to estimate the extent of Nrf2 nuclear translocation (which is indicative of Nrf2 activation) in RAW264.7 macrophages (left panel). Data are expressed as % control by normalizing relative to the vehicle-incubated control group. (B) Following 2 h of-exposure to Sch A or Sch B, the mRNA level of thioredoxin (TRX) was measured, as described in Fig 6. (C) Following a 16 h-exposure to Sch A and Sch B, the level of TRX was measured using Western blot analysis. The amount of TRX was normalized relative to the β-actin level and expressed as % control (right; lower panel). Value given are means ± SEM, with n = 3. * Significantly different from the vehicle control.
Table 3.
Effect of Sch B incubation on lipopolysaccharide (LPS)-activated pro-inflammatory cytokines at 16 h- post Sch exposure in RAW264.7 macrophages.
Fig 8.
Effect of Sch B incubation on lipopolysaccharide (LPS)-activated pro-inflammatory effectors at 16 h-post exposure in RAW264.7 macrophages.
No significant change was indicated as No Sig. Change; No significant suppression was indicated as No Sig. suppressAfter incubation with Sch A or Sch B for 6 h, RAW264.7 macrophages were further incubated with tested compound-free medium for 16 h for the induction of thioredoxin. Cells were then exposed to LPS for 2 h. (A, B) RNA of the cell lysate was isolated and cDNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2) were measured by real time polymerase chain reaction using TaqMan Array plates on the 7500 Fast system. The data were quantified as described in Fig 5. Data were expressed as % non-LPS control by normalizing with the value of non-LPS stimulated cells (Upper panel). (C, D) After exposure to LPS for 18 hours, the level of iNOS and COX2 was measured using Western blot analysis. The amount of iNOS and COX2 were normalized with respective β-actin level and expressed as % non-LPS control (middle panel). (E, F) After exposure to LPS for 24 h, the levels of nitric oxide (NO, the product of iNOS-catalyzed reaction) and prostaglandin E2 (PGE2, the product of COX2-catalyzed reaction) were measured by Griess assay and ELISA, respectively. Data were expressed as % non-LPS control by normalizing with the value of non-LPS stimulated cells (lower panel). Value given are means ± SEM, with n = 3–5. * Significantly different from the non-LPS control; # significantly different from the LPS control.
Table 4.
Effects of CYP inhibitor and Nrf2 knock down on the anti-inflammatory action afforded by Sch B in lipopolysaccharide (LPS)-activated RAW264.7 macrophages.
Fig 9.
Effect of Sch A and Sch B incubation on levels of GSH and interleukin 2 (IL-2) and cell proliferation in concanavalin A (Con A)-activated splenocytes.
Splenocytes were isolated from the spleens of female ICR mice as described in Materials and Methods. Isolated splenocytes were exposed to Sch A or Sch B [alone (25 and 50 μM) or in combination (25 μM)] for 6 h. (A) Cellular GSH levels were measured. Data are expressed as nmol/mg protein (left; upper panel). * Significantly different from the control group. The incubated cells were then stimulated with concanavalin A (4 μg/mL). (B) At 24 h after exposure to Con A, levels of IL-2 (in the culture medium) were measured using ELISA. Data are expressed in pg/mL (left; lower panel). Values given are means ± SEM, with n = 3–5. * Significantly different from the control group; # significantly different from the control group with LPS stimulation. (C) At 72 h after exposure to Con A (1, 2 and 4 μg/mL), the Con A-induced proliferation of isolated lymphocytes was measured using the MTT assay, as described in Materials and Methods. The area under the curve (AUC) plotting the absorbance at 570 nm against concentration of Con A was calculated and expressed as stimulation index. Data are expressed as % control by normalizing relative to the vehicle control group (right panel). Values given are means ± SEM, with n = 3–5. * Significantly different from the vehicle control group.
Fig 10.
Effects of acute/long-term treatment with Sch A and Sch B in carrageenan-induced paw edema in ICR mice.
(A, B) Female ICR mice were administered Sch A or Sch B [single oral bolus dose: alone (1 mmol/kg) or in combination (0.5 mmol/kg); long-term treatment: alone (0.25 nmol/kg/d) or in combination (0.125 nmol/kg/d) × 5 d], as described in Materials and Methods. The left hind limb of the Sch A/Sch Btreated mice was injected with carrageenan (50 μL, 1%, w/v in sterile saline). Time-dependent changes in the thickness of the paw were monitored (upper panel). The area under the curve (AUC) was calculated by plotting % initial thickness of paw against time (lower panel of A and B). Data are expressed as % control by normalizing with the value of vehicle control group. Value given are means ± SEM, with n = 5. * Significantly different from the vehicle control group.
Fig 11.
Effect of acute treatment with Sch A and Sch B on the level of GSH in lymphocytes isolated from ICR mice.
Female ICR mice were administered Sch A or Sch B [single oral bolus dose: alone (1 mmol/kg) or in combination (0.5 mmol/kg)], as described in Materials and Methods. One h after the Sch A/Sch B treatment, heparinized blood samples were drawn from ketamine-anesthetized mice by cardiac puncture. Lymphocytes were isolated by centrifugation in Histopaque 1083, as described in Materials and Methods. Lymphocytes were washed with phosphate-buffered saline (PBS) and lysed with 3% SSA. GSH levels of isolated lymphocytes were measured. Value given are means ± SEM, with n = 5–6. * Significantly different from the vehicle control group.
Table 5.
Effect of the acute treatment with Sch A and Sch B on pro-inflammatory cytokines in carrageenan-induced paw edema in mice.
Table 6.
Effect of the long-term treatment with Sch A and Sch B on pro-inflammatory cytokines in carrageenan-induced paw edema in mice.
Fig 12.
Effects of long-term treatment with Sch A and Sch B in concanavalin A (Con A)-activated splenocytes isolated from Sch A/Sch B-treated mice.
Female ICR mice were administered Sch A or Sch B orally [alone (0.25 nmol/kg/d) or in combination (0.125 nmol/kg/d) × 5 d], as described in Materials and Methods. Splenocytes were isolated from the spleen of Sch A/Sch B-treated mice, as described in Materials and Methods. (A) The isolated splenocytes were lysed and the levels of GSH in the cell lysate were measured. (B) Isolated splenocytes were incubated with Con A (1, 2 and 4 μg/mL) for 72 h. The Con A-induced proliferation of splenocytes was measured by MTT assay. The area under the curve (AUC) plotting the absorbance at 570 nm against the concentration of Con A was calculated and expressed as stimulation index. Data are expressed % control by normalizing with the vehicle control group. * Significantly different from the control group.
Table 7.
Effect of long-term treatment with Sch A and Sch B on T helper cell 1 cytokines in concanavalin A (Con A) -activated splenocytes isolated from Sch-treated mice.
Fig 13.
A proposed mechanism underlying the anti-inflammatory actions of Sch A and Sch B.
Both Sch A and Sch B can elicit a direct anti-inflammatory action via the activation of GST and depletion of GSH, with the resultant suppression of a pro-inflammatory signaling cascade and pro-inflammatory cytokines as well as effectors in LPS-activated macrophages. Sch B can also induce an indirect anti-inflammatory response via the induction of Nrf2-mediated expression of TRX in LPS-activated macrophages. Sch A and Sch B can suppress the proliferation of Th1 cells (which promotes inflammation) via the depletion of GSH and a reduction in the release of Th1 cytokines in Con A-activated T lymphocytes. Abbreviations: Sch A, schisandrin A; Sch B, schisandrin B; GST, glutathione S-transferase; GSH, reduced glutathione; LPS, lipopolysaccharide; JNK, c-Jun N-terminal kinase; p38, p38 kinase; NF-κB, nuclear factor κB; Nrf2, nuclear factor erythroid 2-related factor 2; TRX, thioredoxin; Con A, concanavalin A; Th1, T helper1 subtype.