Reagents and Chemicals
GLGZG was presented by Pharmaceutical Department of Fujian University of Traditional Chinese Medicine Affiliated Second People's Hospital (Fuzhou, China). It was certified and standardized on the basis of labeled compounds (The Food and Drug Administration in Fujian Province 2013). Our phytochemical studies also illustrated that 104 compounds in GLGZG were identified or tentatively characterized and several bioactive components, such as citrulline, luteolin, puerarin, liquiritin, taxifolin, naringin, formononetin, isoliquiritigenin, 6-gingerol, curcumin, caffeic acid, ferulic acid, jujuboside A, protocatechuic acid, cinnamic acid, catechin, paeoniflorin [22,29–31 ]. Lipopolysaccharide (LPS) was purchased from Sigma (St. Louis, USA). RPMI Medium 1640 basic and fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). CellTiter 96® AQ ueous one solution cell proliferation assay was bought from Promega (Madison, USA). IL-1β, IL-6, IL-10 and TNF-α mouse ELISA kits were obtained from (ABclonal (Wuhan) biotechnology co. LTD, Wuhan, China). Rabbit antibodies against Akt, p-Akt(Ser473), PI3K(p85), PI3K(p110α), IKKβ, p-IκBα, IκBα, NF-κBp65 and β-actin were purchased from Cell Signaling Technology (Boston, MA, USA). The secondary antibodies conjugated with horseradish peroxidase (HRP) were all bought from Xiamen LuLong Biotech Co., Ltd. (Xiamen, China).
Cell Culture And Treatment
BV-2 microglial cells and neuronal HT22 were obtained from Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China). In brief, cells were cultured in RPMI 1640 medium with 10% FBS and 1% penicillin-streptomycin mixed solution (Gibco Invitrogen Corporation, Carlsbad, CA, USA) at 37 °C in a 5% CO2 incubator.
In these studies, the cells were seeded on 96-well plates or 6-well plates and were divided into three groups randomly: (1) pure BV2 cell group as control group (BV2 group); (2) LPS group, where BV2 cells were incubated with LPS (1 µg/mL) for 24 h; and (3) LPS plus GLGZG (50,100༌200 µg/mL) group, where cells were co-incubated with LPS (1 µg/mL) and GLGZG (50, 100, 200 µg/mL) for 24 h. Culture supernatants were harvested for ELISA experiments and nitric oxide assay. Additionally, the cells were then harvested for RNA or protein isolation.
To determine protective effect of GLGZG against microglial-mediated neurotoxicity, neuronal HT22 were incubated in conditioned medium from the control, LPS, and LPS plus GLGZG groups of BV2 cells. Conditioned media were then tested for cell viability using CellTiter 96® AQueous one solution cell proliferation and LDH assays, and cell lysates were used to test for apoptosis from conditioned media-treated HT-22 cells.
Cell Viability Assay
After treatment, the cell viability was assessed by CellTiter 96® AQueous one solution cell proliferation assay. CellTiter 96® AQueous one solution was added and incubated for another 4 h at 37 °C. Then the absorbance at 570 nm was taken by a microplate reader (Infinite M200 Pro, TECAN). The cell viability was calculated according to the OD value. Survival rate (%) = ODexperimental group/ODcontrol group ×100%).
Elisa Experiments
After treatment, culture supernatants were collected and then centrifuged prior to the determination of IL-1β, IL-6, IL-10 and TNF-α production. Detailed manipulation process was performed by manufacturer protocols of mouse ELISA kits.
Nitric Oxide Assay
After treatment, the culture supernatants were collected, and NO production was measured by assessing the nitrite level in the culture media. It was executed by mixing the medium with griess reagent. Optical concentration was analyzed at 540 nm after 10 minutes incubation.
Quantitative Real-time Pcr Analysis
After treatment, cells were washed with PBS, and the total RNA was extracted by RNeasy® Mini Kit, and the RNA concentration were determined. Then Revert Aid First strand cDNA Synthsis Kit was used to reverse transcription to cDNA. The cDNA product was used as a template for quantitative PCR amplification, and it was then carried out on ABI 7900HT real-time PCR system (Applied Biosystems, Inc., Foster City, CA, USA). The data were analyzed by 2−△△CT relative quantification method. The relative transcriptional level of target genes normalized to GAPDH was calculated. The primer sequences for amplification of the target genes are shown in Table 1. The relative transcriptional level of target genes normalized to GAPDH was calculated.
Table 1
Primers used for quantitative real-time PCR analysis
Gene | Forward primer | Reverse primer |
CD32 | 5′-AATCCTGCCGTTCCTACTGATC-3′ | 5′-GTGTCACCGTGTCTTCCTTGAG-3′ |
CD86 | 5′-GACCGTTGTGTGTGTTCTGG-3′ | 5′- GATGAGCAGCATCACAAGGA − 3′ |
CD206 | 5′-CAAGGAAGGTTGGCATTTGT-3′ | 5′-CCTTTCAGTCCTTTGCAAGC-3′ |
Arginase1 | 5′-TCACCTGAGCTTTGATGTCG-3′ | 5′-CTGAAAGGAGCCCTGTCTTG-3′ |
Ym1 | 5′-CAGGGTAATGAGTGGGTTGG-3′ | 5′-CACGGCACCTCCTAAATTGT-3′ |
IL-1β | 5′-ATG ACC TGT TCT TTG AGG CTG AC-3′ | 5′-CGA GAT GCT GCT GTG AGA TTT G-3′ |
IL-6 | 5′-GAC CAA GAC CAT CCA ACT CAT C-3′ | 5′-ACA TTC ATA TTG CCA GTT CTT CGT A-3′ |
IL-10 | 5′-CCAAGCCTTATCGGAAATGA-3′ | 5′- TTTTCACAGGGGAGAAATCG-3′ |
TNF-α | 5′-ATG AGC ACG GAA AGC ATG-3′ | 5′-TAC GGG CTT GTC ACT CGA GTT-3′ |
iNOS | 5′-CAA GCA CCT TGG AAG AGG AG-3′ | 5′-AAG GCC AAA CAC AGC ATA CC-3′ |
GAPDH | 5′- AGC CCA GAA CAT CAT CCC TG-3′ | 5′- AGC CCA GAA CAT CAT CCC TG-3′ |
Western Blot Analysis
Western Blot Analysis
The cells were inoculated into 6-well plate with a density of 3 × 105/well and cultured for 24 h, then treated with indicated concentrations of GLGZG and LPS (1 µg/mL) for 24 h. Cells were collected and lysed by lysis buffer, then they were centrifuged at 12,000 g for 15 min. The supernatant was collected and the protein concentration was determined by the BCA method. Then protein mixed with loading buffer and incubated in 100 °C for 6 min. Ultimately, samples were analyzed for western blot analysis with primary antibodies to iNOS(1:1000), CD206(1:1000), p-AKT(Ser473)(1:2000), AKT(1:1000), PI3K(p85)(1:1000), PI3K(p110α)(1:1000), IKKβ(1:1000), p-IκBα(1:1000), IκBα(1:1000), NF-κBp65(1:1000), Bax(1:1000), Bcl-2(1:1000), NeuN(1:1000) and β-actin(1:1000) overnight at 4 ℃ followed by incubating with the horseradish peroxidase-conjugated secondary antibody IgG (HRP Goat Anti-Rabbit IgG secondary antibody (1:5000), Goat Anti-Mous IgG secondary antibody (1:5000), LuLong Biotech Co., Xiamen, China) at room temperature. Finally they were evaluated using the ECL western detection reagents and the relative expression level of target genes normalized to β-actin was analyzed.
Immunofluorescence Assay
Immunofluorescence assay was performed to detect the nuclear translocation of NF-kBp65 subunit. In brief, cells were fixed using 4% paraformaldehyde for 15 min at room temperature, followed by washing with PBS for 5 min 3 times. The cells were blocked with 5% BSA prepared in 0.1% Tween 20 (PBST) and 0.3% Triton X-100 for 1 h at room temperature. Afterward, cells were incubated with NF-κBp65 specific primary antibody (1:100, Cell Signaling Technology, MA, USA) overnight at 4 °C. After washing, cells were incubated with FITC-labeled IgG secondary antibody (Beijing Zhongshan Jinqiao Biotechnology Co. LTD, Beijing, China, 1:200 dilution in 5% BSA solution) for 1 h in dark. After that, cells were stained with 25 µg/mL of 4’-6-diamidino-2-phenylindole (DAPI) in PBST. Finally, samples were documented using Olympus IX73 fluorescence microscope (Tokyo, Japan).
Statistical analysis
All the experiments were repeated three times. The data were statistically analyzed by SPSS 19.0 software and expressed as mean ± standard deviation (`x ± s). The difference between groups was analyzed by one-way ANOVA followed by the Student-Newman-Keuls q test with post-hoc correction was employed for the multiple comparisons, and P < 0.05 was considered to indicate statistical significance.