Materials
TMAO was purchased from Sigma-Aldrich (#317594, USA), and oxidized low-density lipoprotein (ox-LDL) was purchased from Yi yuan biotechnology (YB-002, Guangzhou, China). Phorbol-12-myristate-13-acetate (PMA) was bought from Sigma (USA). Primary antibodies: pro-caspase-1(1:1000, ab207802) 、IL-18(1:500, ab207324)、NF-kb(1:5000, ab76311)、pro-IL-1b(1:1000,ab216995)、β-actin(ab8226,1:10000) and GAPDH(1:5000, ab181602) were bought from Abcam(USA) ; NLRP3(1:700, D4D8T) was purchased from CST(USA); ASC(1:1000, WL02462)、IL-1b mature(1:500, WL00891)、caspase-1p20(1:1000, WL02996a)、p-NF-KB(1:1000, WL02169)、GRP78/Bip(1:1000, WL03157)、p-PERK(1:1000, WL05295) were purchased from Wan lei Bio (Shenyang, China). While secondary antibodies: Goat Anti-Rabbit IgG HRP conjugate(1:10000, S0001, Affinity,Jiangsu),Goat Anti-Mouse IgG HRP conjugate (1:10000, Abcam,ab205719,USA). NLRP3 inhibitor MCC950(HY-12815A,25uM,4h)and NF-KB inhibitor JSH-23(HY-13982,30uM, 1h)were purchased from MCE (Shanghai, China), ER stress inhibitor 4-PBA (SML0309, 5mM,30min) was obtained from Sigma ( USA).
Cell culture and treat
THP-1 cells were grown in RPMI1640 with 10% fetal bovine serum (FBS) and then incubated at 37°C in a CO2 incubator. Cells with suspension state in the logarithmic growth phase were transferred to 6-well or 96-well plates and then were induced to differentiate into adherent M0 macrophages by stimulation with 100 ng/mL PMA for 48 h. Cells were separated into several groups at random, including the TMAO-induced group, ox-LDL-induced group, ox-LDL added TMAO group, MCC950 group, JSH-23 group, and 4-PBA group. The TMAO-induced group and the ox-LDL-induced group were used to determine the dosage of TMAO and ox-LDL.
Cell viability assay
THP-1 cells in the logarithmic growth phase were sown in 96-well plates, and 100μl cell suspension with 2% serum and PMA was added to per well.10μL CCK-8(C0005, Target MOI) solution was subsequently added per well in the dark and measured the absorbance at 450nm wavelength after being cultured for 1-2 h.
Detection of LDH release
After the respective treatment of cells, the supernatant was collected, the appropriate LDH reagent was applied to each well in accordance with the manufacturer's instructions (A020-2-2, Jiancheng Biology, Nanjing), and absorbance was measured at 450 nm wavelength.
Western blot (WB)analysis
Cells were lysed using RIPA lysis buffer (Beyotime, P0013B, China) containing protease inhibitor PMSF (Solarbio, P0100, China) and phosphatase inhibitor (BL615A, Bio sharp, China) after treatment, BCA protein detection kit (Solarbio, China) was used to measure the protein concentration per samples after collecting. Polyvinylidene fluoride (PVDF) membranes were used to transfer the proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separated them (ISEQ00010, Merck). After being blocked with 5% nonfat dry milk, the PVDF membranes was incubated in the appropriate primary antibody solution at 4° for an overnight period, rinsed with PBST, and then incubated with the corresponding secondary antibody for an hour at room temperature. After adding the ECL luminescent agent(P10100,NCM), exposure and development were conducted, and the quantitative analysis of gray value was performed by using ImageJ software.
Enzyme-linked immunosorbent assay (ELISA)
The levels of IL-1β and IL-18 were measured according to the manufacturer's instructions of ELISA kit (JLC6380, JLC6382, Jingkang Biological, Shanghai) after being handled appropriately and harvested supernatants.
Caspase-1 activity detection
Caspase-1 activity assay kit (C1102,Beyotime, Shanghai) was used to measure the activity levels of caspase-1 This assay was based on the ability of caspase-1 to catalytic acetyl-Tyr-Val-Ala-Asp p-nitroaniline (Ac-YVAD-pNA) to the yellow formazan product p-nitroaniline (pNA) .A standard curve for pNA was drawn by measuring the OD values in 405nm. The production of pNA in the test sample indicates the activation level of caspase 1.
Reverse transcription-quantitative PCR (qRT-PCR):
Use of the Trizol reagent extracted total RNA from the THP-1 cells. The reverse transcription kit (RR036A,Takara, Harbin, China) was used to synthesize cDNA, and the target gene was amplified by the SYBER Green method(RR820A,Takara, Harbin, China).NLRP3-related primers came from the remaining in our research group, Bip and PERK primers sequences were designed from Rui Biotech (Beijing, China). The relative expression of the target gene was shown as CT value, the targeting gene's mRNA level was standardized to GAPDH, and calculated by the formula x=2-ΔΔCT (Table 1).
Table 1: primer sequences of gene
Target gene
|
primer sequences
|
GAPDH
|
F
|
TCGGAGTCAACGGATTTGGT
|
R
|
TCGGAGTCAACGGATTTGGT
|
IL-18
|
F
|
ACTGGTTCAGCAGCCATCTT
|
R
|
TGCAGTCTACACAGCTTCGG
|
IL-1b
|
F
|
CCTGCAGCTGGAGAGTGTG
|
R
|
TGTGCTCTGCTTGTGAGGTGC
|
NLRP3
|
F
|
CTTCCTTTCCAGTTTGCTGC
|
R
|
TCTCGCAGTCCACTTCCTTT
|
Caspase-1
|
F
|
GCCCAAGTTTGAAGGACAAA
|
R
|
GGTGTGGAAGAGCAGAAAGC
|
GRP78/Bip
|
F
|
GAACGTCTGATTGGCGATGC
|
R
|
TCTTTGGTTGCTTGGCGTTG
|
PERK
|
F
|
CCAGTTTTGTACTCCAATTGCA
|
R
|
CAGATACAGCTGGCCTCTATAC
|
Statistical analysis
GraphPad 9.0 software was used for statistical analysis, and all data were reported as mean ± SD. A t-test or one-way analysis was utilized to analysis differences, which were deemed statistically significant when p < 0.05.