Hsiao-Wei Chena,1, Tsung-Shi Yangb,1, Mao-Jing Chena, Yu-Ching Changa,Eugene I.-Chen Wangc, Chen-Lung Hoc, Ying-Jang Laid, Chi-Cheng Yue,Ju-Ching Choub, Louis Kuo-Ping Chaob,∗, Pei-Chun Liaob,∗
aChemistry and Environment Laboratory, Taiwan Power Research Institute, New Taipei City, Taiwan
bDepartment of Cosmeceutics, China Medical University, Taichung, Taiwan
cDivision of Wood Cellulose, Taiwan Forestry Research Institute, Taipei, Taiwan
dDepartment of Food Science, National Quemoy University, Kinmen, Taiwan
eGreenlink Biotech Inc., Taipei, Taiwana
In this study, flue gas from a power plant smokestack was applied to culture Spirulina platensis microalgae. Our results will not only achieve the fixation of carbon from the emissions, products can also be producedfrom the algal biomass that possess physiological activities which could be beneficial to human health. An improved one-step process of chromatography was used to produce high-purity C-phycocyanin witha PC ratios >3.5. Adding different concentrations of ammonium sulfate produced different amountsof C-phycocyanin, with 40% generating the highest yield, followed by 35% and 30% concentrations.Immunomodulating activities were evaluated in the murine macrophage cell line J774A.1. We found that C-phycocyanin had the capability to induce secretion of inflammatory cytokines, including TNF-α, IL-1 ,and IL-6, and that these results were not due to contamination with LPS. Treatment with C-phycocyaninalso increased proIL-1 and COX-2 protein expression dose-dependently. Furthermore, C-phycocyaninrapidly stimulated phosphorylation of inflammatory-related signaling molecules, including ERK, JNK, p38and I B. In addition, although C-phycocyanin decreased production of LPS-induced ROS, it did not inhibitLPS-induced inflammatory cytokines in J774A.1 cells. This is the first report to show that C-phycocyanin exhibited a detailed molecular mechanism of bioactivity by boosting immunomodulation performance.
Keywords:C-phycocyanin， Immunomodulating activity，Spirulina platensisa
Because of the excessive discharge of global CO2emissions,we are facing an ever more serious greenhouse effect and otherenvironmental problems. In addition to limiting CO2emissionswithin reasonable constraints, the conversion of carbon sourcesinto usable chemicals or sustainable energy sources is becominga focus of the utilization of global biomass resources. In our previ-ous study which is using flue gas from a power plant smokestackto mass-culture Spirulina platensis microalgae in a photobioreactor . This will not only achieve the fixation of carbon from the emis-sions products can also be produced from the algal biomass thatpossess physiological activities which could be beneficial to humanhealth.Since ancient times, S. platensis has been utilized as a food orfood supplement for humans. In recent years, there has been a trendtoward using the algal products in functional food items or as rawmaterials for cosmetic products. Phycocyanin, the active compo-nent of S. platensis, is a colorant present in cyanobacteria and redalgae. Upon purification, phycocyanins show a brilliant blue colorin solution. Phycocyanins are composed of two subunits and combined together [2–4]. In nature, it exists as monomers, trimersor hexamers; small quantities of oligomers have been found as well. In general, phycocyanins include C-phycocyanin and allophy-cocyanin. They possess different maximum absorption peaks, 620and 650 nm, respectively. Studies have suggested that the ratio of absorbance at 620 and 280 nm can be employed to indicate thepurity of C-phycocyanin, while the ratio of absorbance at 650 and280 nm can indicate the purity of allophycocyanin .In addition, because of their special bioactivities they areincreasingly recognized as potential raw materials for making foodproducts that are beneficial to health . Many studies have indi-cated that phycocyanins have bioactivities that include anti-tumor,antioxidant, and anti-inflammatory effects [8–13]. Several earlierstudies indicated that C-phycocyanin has antioxidant and anti-inflammatory efficacies [12,14–16]. Other reports indicated thatC-phycocyanin has anticancer bioactivity [17,18]. In 2003, a studyby Reddy et al. demonstrated that C-phycocyanin could reducethe proliferation of macrophages (RAW 264.7) with increasingdosages . In addition, C-phycocyanin was found to be able toselectively suppress COX-2 and PGE2expression when RAW 264.7macrophages were stimulated by lipopolysaccharides (LPS), thusdemonstrating the anti-inflammatory capability of C-phycocyanin.Research on immunomodulation by natural products is a con-cern for anti-infective therapies and health care. It is well-knownthat macrophages play an important role in first-line immunologi-cal defense against host infections in mammals, and destroy tumorcells through the secretion of various cytokines . In an immuneresponse, the macrophages directly destroy foreign microorgan-isms and tumor cells by secreting a variety of cytokine mediators,such as interleukin-1 (IL-1 ), tumor necrosis factor- (TNF-α),interleukin-6 (IL-6), etc. IL-1 can prompt the proliferation of T-cells, induce B-cells to produce antibodies, and boost the cytotoxicfunctions of CTL (cytolytic T lymphocytes) and NK (natural killer)cells. IL-6, on the other hand, stimulates the liver to produce acute-phase proteins, and stimulates B-cell proliferation; whereas TNF-αhas direct cytotoxic and growth-suppression effects against tumorcells [20–22].Several obstacles exist in the purification process of C-phycocyanin [23–25]. First of all, when algal yield is high, theC-phycocyanin purity tends to be low. However, if high purityC-phycocyanin is desired, the experimental procedure is rathercomplicated and entails a high production cost. Some studies havebeen carried out with phycocyanin, but there has often been a lackof elucidation of the cell molecular mechanism [26,27]. Thus, themain focus of the present study was on rapid purification of phy-cocyanin from S. platensis and analysis of its bioactivities.
2. MATERIALS AND METHODS
2.1. Chemicals and antibodies
LPS (from E. coli 0111:B4), polymyxin B (PMB), monoclonal anti-MAP kinase,activated (diphosphorylated ERK) antibody, monoclonal anti-JNK kinase, activated(diphosphorylated JNK) antibody, monoclonal anti-p38 MAP kinase, activated(diphosphorylated p38) antibody, monoclonal anti-actin antibody and commer-cial C-phycocyanin were purchased from Sigma–Aldrich (St. Louis, MO, USA). Amouse IL-1 , TNF-α and IL-6 ELISA (enzyme-linked immunosorbent assay) kitwas purchased from R&D Systems (Minneapolis, MN, USA). Anti-IL-1 polyclonalantibody, anti-I B- antibody, anti-phospho-I B- antibody, anti-COX-2 antibody,anti-rabbit IgG-HRP, and anti-mouse IgG-HRP were obtained from Santa CruzBiotechnology (Santa Cruz, CA, USA).
2.2. Extraction of crude phycocyanin from S. platensis
S. platensis microalgae were cultured in the three-dimensional photobioreactorat the Dalin power plant in southern Taiwan . A simple repetitive freeze-and-thawprocess was applied to degrade the cell walls of S. platensis microalgae.
2.3. Purification of pure C-phycocyanin from crude phycocyanin
Then, using ultrasonic vibratory treatment in PBS, crude C-phycocyanin was dissolved. In brief, 20 g (oven-dried) of S. platensis biomass was soaked at room temperature in 300 ml of PBS (1 L of PBS contained 137 mmol of NaCl, 2.7 mmol of KCl, 10 mmol of Na2HPO4, and 1.76 mmol of KH2PO4; pH = 7.4); then the solutionwas placed in an ultrasonic bath (Branson 5510), sonicated and extracted for 2 h. Thesolution was placed in a −80◦C freezer for 24 h, then deiced at room temperatureand centrifuged using an ultracentrifuge at 4◦C (12,000 rpm, 30 min). To the super-natant, a series of (NH4)2SO4(30, 35 and 40%) were added separately to salt out the protein. After repeating 4◦C ultracentrifugation (12,000 rpm, 30 min), the pre-cipitated protein fraction was re-dissolved with PBS and filtered through a 0.22 mmembrane. Dialysis was carried out using a Spectra/Por®membrane (Spectrum Lab-oratories, Rancho Dominguez, CA, USA) with a molecular weight cutoff of 1 kDa.Water was replaced with fresh deionized water twice daily for 2 days. The resultingC-phycocyanin was then freeze-dried and quantified. Two hundred mg of crude C-phycocyanin (PC ratios ca. 0.8) was weighed and redissolved in PBS. Then a GPC 1122liquid chromatography solvent delivery system (LC Tech, Dorfen, Germany) fittedwith a HiPrep 26/60 SephacrylTMS-300 high-resolution column (GE Healthcare Bio-Sciences, Uppsala, Sweden) was used to purify C-phycocyanin. The conditions ofisolation were: each injection amount was 5 ml, elution solution was 1 × PBS (con-taining 0.05% NaN3), the flow rate of eluent was set at 3.0 ml/min, and 260 s per tubewas collected. C-phycocyanin from the fifth tube on a UV-vis spectrophotometer(Agilent 8453; Agilent Technologies, Santa Clara, CA, USA) was used to determinethe PC ratios (A620/A280) of the contents. For PC >2.0, the fraction was separately collected, then dialysis was repeated (Spectra/Por®membrane, molecular weightcutoff 1 kDa); every 12 h the water was replaced. After three exchanges, the post-dialysis phycocyanin was freeze-dried, collected separately and quantified. In orderto increase the separation efficiency, the NaCl concentration gradient of the eluentadded was from 0 mM to 30 mM.
2.4. Cell cultures
J774A.1 murine macrophages obtained from the Bioresource Collection andResearch Center (Taiwan, ROC) were propagated in RPMI 1640 medium supple-mented with 10% heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT,USA) and 2 mM L-glutamine (Invitrogen Life Technologies, Carlsbad, CA, USA), andcultured at 37◦C in a 5% CO2incubator.
2.5. Cell toxicity of C-phycocyanin
Cell proliferation was determined using an MTT assay . J774A.1 macrophageswere seeded in 96-well plates at a density of 5 × 103 cells/well. Cells were incu-bated with C-phycocyanin (50–400 μg/ml) for 24 h. The value was determined byaveraging triplicate sample measurements.
2.6. Western blotting analysis
Whole cell lysates or C-phycocyanin samples were separated by 12% SDS-PAGEand electrotransferred to a polyvinylidene fluoride (PVDF) membrane. The mem-brane was incubated in blocking solution (5% nonfat milk in PBS with 0.1% Tween20) at room temperature for 1 h, and then incubated at room temperature for 2 h withanti-proIL-1 antibody, anti-MAP kinase antibody, diphosphorylated ERK-1 and -2antibody, anti-JNK kinase antibody, anti-p38 MAP kinase antibody, anti-COX-2 anti-body, or anti-I B antibody. After washing three times in PBS with 0.1% Tween 20,the membrane was incubated with an HRP-conjugated secondary antibody directedagainst the primary antibody. The membrane was developed by an enhanced chemi-luminescence Western blotting detection system (DuPont NEN Research Products,Boston, MA, USA) according to the manufacturer’s instructions .
2.7. Enzyme-linked immunosorbent assay (ELISA)
In the dose response study, J774A.1 cells (1 × 106/ml) were stimulated with C-phycocyanin only or with C-phycocyanin and LPS for determining TNF-α andIL-6 (after 6 h) and IL-1β (after 24 h). The concentration of cytokines in the con-ditioned medium was analyzed by ELISA according to the manufacturer’s protocol,using Quantikine®mouse TNF-α, IL-6 and IL-1β immunoassay kits (R&D Systems). Biotinylated antibody reagent (50μL) and 50μL supernatant concentrate fromsamples tested for various times were added to anti-mouse TNF-α, IL-6 and IL-1β precoated stripwell plates, followed by incubation at room temperature for 2 h. Afterwashing the plates three times with the washing buffer provided, 100μL dilutedstreptavidin-HRP concentrate was added to each well and incubated at room tem-perature for 30 min. The washing process was repeated; then 100μL premixed TMBsubstrate solution was added to each well and developed at room temperature inthe dark for 30 min. Following the addition of 100μL provided stop solution to eachwell to stop the reaction, the absorbance of the plates was measured by a Gem-ini EM microplate spectrofluorometer (Molecular Devices, Sunnyvale, CA, USA) at 450–550 nm wave lengths.
2.8. Monitoring of LPS contamination of C-phycocyanin in experimentsPrevious studies of C-phycocyanin-mediated reactions and signaling haveencountered the problem of LPS contamination. Reagents and utensils for thepreparation of C-phycocyanin were either LPS-free grade or were washed withPBS containing 10μg/ml PMB, then rinsed with PBS. In order to rule out possi-ble LPS contamination of C-phycocyanin samples, J774A.1 cells were pre-incubatedwith or without PMB (10μg/ml) for 30 min, followed by treatment for 6 h withC-phycocyanin (400 μg/ml) or 24 h with LPS (0.03, 0.1, 0.3 or 1μg/ml).
2.9. Antioxidant activity of C-phycocyanin
The antioxidant activity of C-phycocyanin was determined by intracellular H2O2stimulated by C-phycocyanin, which was measured by detecting the fluorescenceintensity of chloromethyl-2',7'-dichlorofluorescin (CM-DCF), the oxidized productof chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCDA) (Molec-ular Probes, Eugene, OR, USA). Briefly, J774A.1 cells (1 × 106/ml) grown in serum-and phenol red-free RPMI medium for 24 h were then preincubated with CM-H2DCDA (2μm) and N-acetyl cysteine (NAC) (10 mM) at 37◦C for 30 min in the dark.This was followed by adding fresh starvation medium containing C-phycocyanin(50–400 μg/ml) or LPS (1μg/ml) for an additional time, as indicated above. Therelative fluorescence intensity of the fluorophore CM-DCF, which was formed byperoxide oxidation of the nonfluorescent precursor, was detected at an excitationwavelength of 485 nm and an emission wavelength of 535 nm with a Gemini EMmicroplate spectrofluorometer (Molecular Devices).
2.10. Statistical analysis
All values are given as mean ±SD. Statistical analysis was performed by ANOVAfor analysis and followed by suitable post hoc Duncan’s multiple range test (DMRT)test was applied to calculate the statistical significance between various groups. Avalue of P < 0.05 was considered to be statistically significant.
3.1. Isolation and purification of C-phycocyanin
A notable feature of the present study was that the purificationof C-phycocyanin was not as complicated as those described in ear-lier studies. The process merely involved dissolving C-phycocyaninand protein from S. platensis using phosphate buffered saline(PBS); then a 40% (NH4)2SO4solution was added to salt out crudeC-phycocyanin. Upon filtration using a 0.22 μm membrane, the products were freeze-dried and quantified. A gel-filtration columnwas used to obtain pure C-phycocyanin with PC ratios >3.5. Theflow diagram of the procedure is shown in Fig. 1.
Table 1 shows that by using different concentrations of ammo-nium sulfate, different yields of C-phycocyanin were obtained. The 40% group produced the highest yield. C-phycocyanin obtained with different ammonium sulfate concentrations were separately collected at PC fraction ratios of 2.5, 3.0, 3.5 and >3.5. The results indicated that the higher ammonium sulfate concentration, the greater C-phycocyanin yield. However, regardless of ammoniumsulfate concentrations, the higher PC ratios of the fraction, the loweryield became. In addition, Table 1 also shows the average precipi-tation yields of C-phycocyanin respectively with 30%, 35%, and 40% ammonium sulfate solution, which were respectively 6.54%, 8.37%,and 15%. The average fractional yields (in mg) of C-phycocyaninwith different PC ratios are shown at the lower right corner of Table 1.
The spectra and absorbance values of different purified C-phycocyanin fractions collected are showed in Fig. 2A. After fraction17, the PC ratios decreased rapidly to less than 2.5. Test specimenscollected after fraction 14 were subjected to SDS electrophoresis(Fig. 2B). In the figure, line 1 was from the standard purchased fromSigma–Aldrich, which had a PC ratios >3.5. Line 2 was our puri-fied C-phycocyanin, having a PC ratio of 3.51. Fig. 2A and B was toprove that the C-phycocyanin obtained in the study was not merelyproven using the PC ratios, but the SDS-PAGE was engaged to provethat the purity of C-phycocyanin was similar to the purchased high-purity product from Sigma Chemicals; that the α and β subunits of C-phycocyanin had identical positions and we have remarked the α and β subunits of C-phycocyanin in Fig. 2B. These experimentalresults indicated that using the experimental procedure producedreliable high-purity C-phycocyanin.
Fig. 2. Purity of C-phycocyanin from Spirulina platensis. (A) UV–vis spectra and PCratios of different fractions of crude C-phycocyanin after liquid chromatographicpurification using a SephacrylTM S-300 high-resolution column. (B) SDS-PAGE ofpurified C-phycocyanin (fraction 14) vs. standard (Sigma–Aldrich), both with PCratios >3.5.
3.2. Cytotoxicity and immunomodulation activity ofC-phycocyanin
Prior to investigating the bioactivity of C-phycocyanin, an MTTassay was used to test for cytotoxicity. The results revealed thatwhen C-phycocyanin (400μg/ml) or LPS (1μg/ml) were added tocells, followed by incubation for 24 h, no cell death occurred. Theparticular dosage of both C-phycocyanin (25–400μg/ml) and LPS had no bearing on cytotoxicity (Fig. 3A).Infection by pathogenic bacteria will cause inflammatory cytokines such as TNF-α, IL-1β and IL-6 or their modulating fac-tors to increase, which in severe cases may lead to the death ofpatients. Previous studies have shown that C-phycocyanin con-tains anti-inflammatory capability in LPS-stimulated RAW 264.7macrophages [15,31]. In order to examine whether C-phycocyaninpurified from S. platensis has anti-inflammatory activity in murine J774A.1 macrophages during an infection process, we first added different doses of C-phycocyanin to macrophages, incubated themfor 30 min, and then added LPS (1μg/ml) followed by incubationfor 6–24 h. Fig. 3B–D shows that in murine macrophages pretreated with C-phycocyanin, there was no significant suppression of the expression of TNF-α, IL-1β and IL-6. In LPS-stimulated cells, the secretion amount of TNF-α was 24 ng/ml. Under pretreatment conditions using 50, 100, 200 and 400μg/ml of C-phycocyanin,however, the ensuing TNF-α was 34, 36, 41 and 47 ng/ml, respec-tively (Fig. 3B). LPS stimulation alone caused IL-6 secretion of 10 ng/ml, whereas with pretreatments of 50, 100, 200 and400μg/ml of C-phycocyanin, the secretion of IL-6 rose to 11, 12,13 and 16 ng/ml (Fig. 3C). From stimulation by LPS alone, IL-1β expression was 130 pg/ml; where as after pretreatment with the aforementioned C-phycocyanin doses, the expression levelsincreased to 131, 135, 139 and 151 pg/ml, respectively (Fig. 3D).We also used western blotting to test protein expression of inflammatory-related proteins, include proIL-1β and COX-2. Asshown in Fig. 3E, J774A.1 macrophages were pretreated with0–400 μg/ml of C-phycocyanin for 30 min; then cells were stim-ulated with 1 μg/ml of LPS, followed by incubation for 6 h. There sults indicated that there was no discernible suppression of theexpression of proIL-1β and COX-2 as stimulated by LPS.
Fig. 3. Cytotoxicity and effects of C-phycocyanin on LPS-stimulated inflammatory mediators. (A) Cell toxicity of f C-phycocyanin (C-PC) was assayed by Alarmar Blue method.(B) and (C) J774A.1 macrophages subjected to treatment with C-phycocyanin (C-PC, 0–400 μg/ml) for 30 min, followed by stimulating with 1 μg/ml of LPS for 6 h. Secretedamount of TNF-α (B), IL-6 (C) in culture medium was assayed by ELISA. (D) Secreted amount of IL-1βfrom J774A.1 macrophages subjected to treatment with C-phycocyanin(C-PC, 0–400 μg/ml) for 30 min, then with 1μg/ml of LPS followed by incubating for 24 h. The cytokine concentration in culture medium was assayed by ELISA method. Dataare expressed as mean ± SE from three separate experiments. (E) J774A.1 macrophages (1 × 106/ml) were pretreated with 0–400μg/ml of C-phycocyanin (C-PC) for 30 min,followed by stimulating with 1μg/ml of LPS for 6 h. The expression of pro-IL1β and COX-2 was assayed by Western blot. The result of one of three separate experiments isshown.
These results differed from those of Cherng et al. , whichshowed that in a RAW 264.7 (murine microphage) cellular model,the presence of C-phycocyanin could significantly suppress theexpressed amount of iNOS and NF- kB activation stimulated byLPS (120μg/ml). Their results indicated that C-phycocyanin hasthe capacity to suppress production of cytokines when murineRAW 264.7 macrophages were stimulated by LPS. Because ourdata differed from a previous study , we next investi-gated whether C-phycocyanin contains other bioactivity, such asimmunomodulation. To examine the immunomodulation activi-ties of C-phycocyanin, J774A.1 macrophages were treated with0–400μg/ml of C-phycocyanin for 6 h; the secretion of cytokinesin the medium was tested using an ELISA method. To exclude thepossibility of LPS contamination, cells were co-treated with PMB,a cyclic amphipathic peptide antibiotic that binds to endotoxins.As shown in Fig. 4A, the secreted amount of TNF-α increased withincreasing amounts of C-phycocyanin. However, even after adding10 g/ml of PMB, the expressed amount of TNF-α boosted by C-phycocyanin was not totally suppressed. Fig. 4B shows the effectsof adding 50–400μg/ml C-phycocyanin on the production of IL-6by the J774.1 cells. The trend was similar to that in Fig. 4A. The effectof C-phycocyanin stimulation of J774.1 macrophages on the expres-sion of IL-1βis shown in Fig. 4C. Notably, at a 50 g/ml dose, therewas no IL-1βproduction; but at 100–400μg/ml C-phycocyanindoses there was significant activation of IL-1βsecretion. Addi-tionally, when only C-phycocyanin (400μg/ml) without LPS wasadded to J774A.1 macrophage culture, the expression of COX-2 wasincreased dose-dependently; while proIL-1β expression was onlydetected after adding a 400μg/ml dose of C-phycocyanin (Fig. 4D).
When added alone, a 400μg/ml dose of C-phycocyanin causedonly a mild expression of TNF-α (Fig. 4A), unlike that induced by ageneral pathogen (antigen) that releases large amounts of inflam-matory cytokines. In other words, the expressed amount was notvery high: less than half of what 0.03μg/ml of LPS could induce(Fig. 4E). Therefore, C-phycocyanin evidently possesses bioactivity,i.e. the ability to modulate immune cells in mammals. Cytokinessuch as IL-1βcould prompt the proliferation of T-cells, induce B-cells to produce antibodies, and boost the cytotoxic functions of CTL (cytolytic T lymphocytes) and NK (natural killer) cells. IL-6stimulates the liver to produce acute-phase proteins and B-cellproliferation; whereas TNF-α has direct cytotoxic and growth-suppression effects against tumor cells.
These results, however, differed somewhat from the findingsof previous reports [15,31]. In their study, the C-phycocyanin was from Sigma–Aldrich and the PC was >3.5. Therefore, in this studya comparison was made of the differences in bioactivities between commercial C-phycocyanin (purchased from Sigma–Aldrich) andC-phycocyanin purified from Spirulina grown in a photobioreac-tor. First of all, the commercial C-phycocyanin was a liquid withfair amount of salts, including EDTA and PBS (155 mM). In addi-tion, after adding commercial C-phycocyanin alone (50μg/ml) theJ774A.1 murine macrophages had only a 75% survival rate (data notshown). If the C-phycocyanin (50μg/ml) was added 30 min before LPS stimulation, the cellular survival rate increased to 89%. Con-versely, when C-phycocyanin purified from Spirulina was added at 400μg/ml dose, there was no cell death. Apparently, without dial-ysis, the commercial C-phycocyanin preparation might be slightlycytotoxic. At the same dose (50μg/ml), however, we found no apparent suppression effect on the production of TNF-α by J774A.1murine macrophages when commercial C-phycocyanin was used(data not shown).
3.3. Monitoring of LPS contamination of C-phycocyanin inexperiments
In order to confirm the immunomodulating effects of C-phycocyanin and ascertain that the purified C-phycocyanin fromS. platensis was not contaminated with LPS, a set of experiments was designed to prove the authenticity of the experimental results.As shown in Fig. 4E, LPS was diluted separately to 0.03, 0.1, 0.3 and 1μg/ml to stimulate the J774A.1 macrophages; then 10μg/ml of PMB (a polypeptide antibiotic with a positive charge, which is often used to suppress the cytokines stimulated by LPS in in vitro studies) was added separately to suppress the expression of the inflamma-tory cytokine TNF-α. The results indicated that 10μg/ml of PMBcould totally suppress the production of TNF-α at LPS doses of 0.03,0.1 and 0.3μg/ml; however, it failed to totally suppress the expres-sion of TNF-α after a 1μg/ml dose of LPS (the expressed amountwas 200 pg/ml). When cells were stimulated by C-phycocyanin(400 g/ml), the expression of TNF-α increased to 300 pg/ml; and after the further addition of 10 μg of PMB, the TNF-α decreased only slightly, to 250 pg/ml. These results clearly show that the TNF-αproduced by C-phycocyanin was affected only minimally by PMB.This may primarily be due to the charged nature of the protein. If the C-phycocyanin contained an extremely low concentration ofLPS, then its existence in the C-phycocyanin must be >0.075%. Evenif this were true, then 10 μg/ml of PMB can totally suppress the expression of TNF-α. Therefore, the experimental results should be construed as proof that our C-phycocyanin was totally free from contamination by LPS, and that the results were a manifestation purely of C-phycocyanin induction on the J774A.1 macrophages.
3.4. Activation of MAPK and NF-kB signaling pathways byC-phycocyanin
MAPKs and NF-kB signaling are involved in cytokine secre-tion and proinflammatory molecule expression in macrophages.A dosage of 400μg/ml of C-phycocyanin applied to J774A.1 cells appeared to activate the mitogen-activated protein kinases(MAPKs) ERK, JNK and p38 by phosphorylation. After 10 and 60 minof treatment, phosphorylation of I kB was detected. As is wellknown, after phosphorylation, I kB will be degraded by protea-somes; this allows NF-kB to enter the nucleus. The results of thisexperiment suggested that C-phycocyanin indeed possesses bioac-tivity, and is able to modulate murine macrophages (Fig. 5).
3.5. Antioxidant activity of C-phycocyaninA previous study has demonstrated that C-phycocyanin containsantioxidant properties . Fig. 6 shows the antioxidant effectsof C-phycocyanin-pretreated J774A.1 cells. LPS stimulation of cellsrapidly induces ROS production when compared with that of con-trol cells. In contrast, pretreatment with N-acetylcysteine (NAc), a
potent antioxidant, quickly reduces the production of LPS-induced ROS. Along with increasing dosages of C-phycocyanin, the ROS(H2O2) content in cells was decreased within 2 h.
Fig. 4. Effect of C-phycocyanin (C-PC) on the expression of inflammatory mediators. (A)–(C) J774A.1 macrophages were treated with C-phycocyanin (C-PC, 0–400μg/ml)for 6 h or 24 h in the absence or presence of 10μg/ml PMB. TNF-α (A), IL-6 (B), IL-1β(C) concentration in culture medium was assayed by ELISA. Data are expressed asmean ± SE from three separate experiments. (D) J774A.1 cells were treated with 0–400μg/ml of C-phycocyanin (C-PC) for 6 h. Cells lysates were separated by 10% SDS–PAGEand immunoblotted with anti-pro-IL1βand anti-COX-2. Shown are representative blots of at least three repeats at all data points. (E) J774A.1 cells were pretreated with LPS(0.03–1μg/ml) or C-phycocyanin (C-PC, 400μg/ml) for 6 h in the absence or presence of 10μg/ml PMB. The TNF-α concentration in culture medium was measured by ELISA.
In Table 1, our results demonstrated that higher concentrationof ammonium sulfate resulted in higher yields of C-phycocyanin of higher PC ratios. Comparing the 40% and 30% ammonium sulfate, the yield gain was less than 2-fold (11.35% vs. 6.54%), however,the fraction of C-phycocyanin with PC ratios >3.5 were 1.75 mgvs. 0.5 mg; for product of PC ratio = 3.5, the yields were 5.0 mg vs. 1.87 mg. Apparently, the high concentration of ammonium sul-fate was an important factor in favoring products of high PC ratios(i.e., produced higher yield and greater purities). This experimen-tal study showed that C-phycocyanin isolated from S. platensismicroalgae possesses immunomodulatory activity. In this study theexperimental results proved that purified C-phycocyanin inducedsecretion of immune cytokines such as TNF-α, IL-1βand IL-6, andregulated intracellular proteins such as proIL-1β, COX-2, phos-phorylated Ikβ, and MAPK in J774A.1 murine macrophages. Inaddition, C-phycocyanin contained antioxidant activity. However,C-phycocyanin produced no apparent anti-inflammatory bioactiv-ity in a J774A.1 murine macrophage model.
In our previous study, a novel photobioreactor was developedwhich is utilizing CO2 in the flue gas of a power plant as the carbonsource for the growth of a seawater alga, S. platensis. This will not only accomplish the fixation of carbon from the emissions; prod-ucts can also be produced from the algal biomass that possesses physiological activities. Otherwise, microbial decomposition will quickly return the fixed carbon back into the atmosphere. Addi-tionally, in the process of treating biomass for reutilization, the necessary energy requirements and ensuing CO2 emissions mustbe taken into account in order to avoid overloading the environ-ment. Nevertheless, the culturing of algal biomass is likely to have a positive overall significance in carbon sequestration. Regardless of whether the biomass is used as a health food supplement or as ani-mal feed, carbon will be fixed in biological entities as a consequence.Therefore, further efforts toward understanding the bioactivities ofsuch biomass are warranted.
Many studies have traditionally used the ratio of absorbance at 620 and 280 nm to express the purity of C-phycocyanin. In a paper by Pinero Estrada et al., a hydroxyapatite column wasfirst used to purify phycocyanin, and then a DEAE Sephadex A-50(Sigma–Aldrich) gel-filtration column to further purify the prod-uct . They obtained a product with an A620/A280 nm ratio of approximately 3.9. Other studies have used an absorbance ratio of A615/A280 nm as an indication of C-phycocyanin purity . How-ever, their reported C-phycocyanin purities were all quite low(>0.5).
Chaiklahan et al. successfully utilized a membrane process to extract phycocyanin en masse from a Spirulina sp. using ultra-filtration. Although the procedure produced larger quantities of phycocyanin, the PC purity value was only ca. 1.07, and thusnot very high [4,33]. Unlike some prior studies, which generally were run at a 50% concentration of ammonium sulfate, relativelylower concentrations were used. In the present study it was found that with increasing ammonium sulfate concentration, the yield ofcrude phycocyanin tended to increase; however, non-target mot-ley proteins of other origin also increased in the solution. Thus,although the total protein extraction increased, the purificationprocess became more complicated as well. As a consequence, a 40% concentration of ammonium sulfate was used to precipitate crudeC-phycocyanin. Ammonium sulfate solutions of 30 and 35% tendedto produce too low a yield, however.
In this study a murine macrophage cell line, J774A.1, was employed as an experimental model. The results showed thatthe C-phycocyanin purified from S. platensis, when applied to amurine J774A.1 macrophage model, exhibited bioactivity by boost-ing immunomodulation performance, and had the capability ofinducing the expression of TNF-α, IL-1β, IL-6, proIL-1 β and COX-2 in macrophage cells, as well as the phosphorylation of ERK, JNK,p38 and IkB. However, the results appeared to deviate from thoseof Cherng et al. . They showed that in a RAW264.7 (murinemicrophage) cellular model, the presence of C-phycocyanin couldsignificantly suppress the expressed amount of iNOS stimulatedby LPS (120μg/ml). In their study, the C-phycocyanin was fromSigma–Aldrich and the PC was >3.5. They also noted that 1 h afterC-phycocyanin treatment at doses of 120–250μg/ml, the expressed amount stimulated by LPS also showed significant suppression;however, C-phycocyanin treatment had no suppressive effect on IL-1β. When LPS was added alone, I kB- was degraded significantly; however, when C-phycocyanin was also added, the degradation could be prevented. Their results indicated that C-phycocyanin has the capacity to suppress production of cytokines when murinemacrophage cells were stimulated by LPS. One possible causeof differences in the experimental results might originate from the source of C-phycocyanin proteins. The C-phycocyanin used intheir study was from Sigma–Aldrich, while in the present studyC-phycocyanin was purified from Spirulina grown in a photobiore-actor. Commercial C-phycocyanin shows slightly cytotoxicity and has no suppression effect on the production of TNF-αby J774A.1murine macrophages (data not shown). In addition, there may beseveral probable causes for the discrepancy, including different cel-lular strains. Cherng et al.  and Reddy et al.  employed RAW264.7 murine macrophages and different LPS. (They applied an E.coli 026:B6 strain, while in the present study an E. coli 0111:B4strain was used.)
Because macrophage cells exist widely in various organs and tissues of the human body, they play a vital role in immunedefense, including participating in immune response and inflam-matory response, and maintaining the stability of the internalcellular environment. However, not all these functions are exe-cuted or expressed by macrophage cells simultaneously. Simplyput, there exists among macrophage cells certain heterogeneity. The probable cause of this phenomenon is that macrophages arederived from different precursor cells which originally are situatedin different growth environments . In addition, different strainsof macrophage cells might differentiate because of their varyinggenetic features . Certain macrophage cells have an expressive nature disassociated from environmental factors. Conversely, they tend to react differently to certain specific stimulus cells. This is because different stimulating factors will cause activation of differ-ent intracellular signal transduction, resulting in macrophage cellheterogeneity.
Several signal transduction cascades are involved in the regu-lation of inflammatory mediator expression, such as MAPKs and NF-kB. Few studies have been conducted on the effects of phy-cocyanins on MAPKs, especially with respect to immunologicalcells. The present results indicated that such cells treated with C-phycocyanin will show activation of ERK, JNK and p38 protein expressions. In a study in a melanoma B16F10 cellular model,adding C-phycocyanin alone could increase the expression of ERK,but at the expense of suppressed p38 expression . Nishanthet al.  found that C-phycocyanin added to hepatoma cells could suppress the content of reactive oxygen species (ROS) and COX-2,but did not affect the expression of ERK, JNK and p38 proteins.ROS play important roles in LPS-mediated cytokine expression.In this study, we found that C-phycocyanin contains anti-oxidantactivity. Our results are consistent with a previous study [9,10],although different cell strains and sources of C-phycocyanin were used. It is generally accepted that ROS has an inflammatory effect,and its over production can cause functional chaos in cells andorgans. A study by Zmijewski et al. , however, indicated that peroxides somehow have an anti-inflammatory effect. Through observation of the suppression of peroxidase or reduction inthe induced inflammatory response by lipopolysaccharides, they explained the mechanism whereby increased peroxides in cellscause acute inflammation. The results of these studies suggest that when the immune system is boosted, it is necessary for ROS in cells to increase as well.
In conclusion, a moderate stimulation of macrophages by C-phycocyanin can increase the immunological activity of cells and allow the biological entity to obtain the benefits. As far as we know,no other paper has studied the intracellular molecular mechanisms of C-phycocyanin activities in such detail.
This work was supported by a grant from the Taiwan PowerResearch Institute, R.O.C.(contract/grant numbers T546CH00007);NSC 96-2116-M039-001-MY3 and NSC 99-2116-M039-001 fromMinistry of Science and Technology, R.O.C.(L.K. Chao) for financialsupport. We also thank Mr. Christopher Salisbury for editing theEnglish in this article.
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