[Mechanism of Tongfu Lifei decoction inhibiting the programmed death-1/programmed death-ligand 1 signaling pathway in THP-1 cells by regulating microRNA-146a].

Abstract

Objective: To explore the protective effect and mechanism of Tongfu Lifei decoction (TFL) on human monocytic leukemia cell THP-1 induced by lipopolysaccharide (LPS).

Methods: (1) THP-1 cells were cultured in vitro, and incubated with 1 mg/L LPS for 18 hours to construct an in vitro THP-1 cell inflammation model. Other THP-1 cells were taken as blank control group. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) secreted by cells. (2) THP-1 cells were divided into seven groups and treated with 0, 0.005, 0.01, 0.02, 0.04, 0.08, and 0.16 mL/mL TFL for 24 hours (added different dosages of TFL solution per milliliter of culture medium, with a crude drug content of 1 kg/L). The cell survival rate was detected using methyl thiazolyl tetrazolium (MTT) colorimetric method, and the intervention dosage of TFL for its non-toxic effect on THP-1 cells was screened. (3) Another THP-1 cells were divide into inflammatory model group and 0.01, 0.02, and 0.04 mL/mL TFL groups according to the intervention dosage of TFL screened by MTT colorimetry. After 24 hours of intervention, the levels of TNF-α and IL-6 secreted by cells were measured using ELISA. Western blotting was used to detect the expressions of programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) signaling pathway proteins in cells. Real time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expressions of microRNAs (miR-146a, miR-146b, miR-155) in cells. (4) The maximum non-toxic concentration of TFL (0.04 mL/mL) on the THP-1 cell was selected as the intervention dose. THP-1 cells were divided into inflammation model group, TFL group, TFL+miR-146a inhibitor group, TFL+miR-146b inhibitor group, and TFL+miR-155 inhibitor group. The inflammation model group was not given any drug intervention. The other inhibitor groups were added 100 nmol/L corresponding inhibitor. After 24 hours of intervention, the levels of TNF-α and IL-6 secreted by cells were measured using ELISA. Western blotting was used to detect the expressions of PD-1/PD-L1 signaling pathway proteins in cells.

Results: (1) Compared with the blank control group, the levels of TNF-α and IL-6 secreted by cells in the inflammatory model group were significantly increased, indicating the successful construction of the THP-1 inflammatory cell model in vitro. (2) 0-0.04 mL/mL TFL had no toxic effect on THP-1 cells. However, the survival rates of cells in the 0.08 mL/mL and 0.16 mL/mL TFL groups were significantly lower than those in the inflammation model group, indicating that TFL dosages exceeding 0.04 mL/mL had toxic effects on THP-1 cells. (3) Compared with the inflammation model group, 0.01 mL/mL TFL had no significant effect on the levels of TNF-α and IL-6 secreted by THP-1 cells, while intervention with 0.02 mL/mL and 0.04 mL/mL TFL significantly reduced the levels of TNF-α and IL-6 secreted by cells [TNF-α(ng/L): 95.89±8.55, 70.73±11.70 vs. 137.10±7.19, IL-6 (ng/L): 23.03±2.55, 16.58±1.72 vs. 32.60±2.55, all P < 0.01]. Compared with the inflammation model group, the expressions of PD-1/PD-L1 signaling pathway proteins in THP-1 cells in different dosages of TFL groups were significantly reduced, and showed a certain dosage dependence. The expressions of the pathway proteins in the 0.04 mL/mL TFL group were significantly lower than those in the inflammation model group [PD-1 protein (PD-1/β-actin): 0.28±0.04 vs. 1.00±0.10, PD-L1 protein (PD-L1/β-actin): 0.54±0.05 vs. 1.00±0.08, phosphoinositide 3-kinase (PI3K) protein (PI3K/β-actin): 0.28±0.03 vs. 1.00±0.08, phosphorylated protein kinase B (p-Akt) protein (p-Akt/Akt): 0.38±0.04 vs. 1.00±0.10, all P < 0.01]. Compared with the inflammation model group, the expression of miR-146a in THP-1 cells in the 0.01, 0.02, and 0.04 mL/mL TFL groups was significantly reduced (2^-ΔΔCt: 0.46±0.11, 0.31±0.13, 0.23±0.14 vs. 1.01±0.18, all P < 0.01), while there was no significant change in the expressions of miR-146b and miR-155. (4) Compared with the inflammation model group, the TFL group showed a significant decrease in the levels of TNF-α and IL-6 secreted by THP-1 cells. The miR-146a inhibitor could significantly reverse the inhibitory effect of TFL on inflammatory factors, and the difference was statistically significant as compared with the TFL group [TNF-α (ng/L): 138.55±10.30 vs. 72.33±10.59, IL-6 (ng/L): 31.35±3.98 vs. 15.75±3.76, both P < 0.01]. Compared with the inflammation model group, the expressions of PD-1/PD-L1 signaling pathway proteins in THP-1 cells in the TFL group were significantly reduced. The expressions of pathway proteins in cells in the TFL+miR-146a inhibitor group were significantly higher than those in the TFL group [PD-1 protein (PD-1/β-actin): 0.85±0.09 vs. 0.37±0.04, PD-L1 protein (PD-L1/β-actin): 0.83±0.08 vs. 0.55±0.06, PI3K protein (PI3K/β-actin): 0.85±0.09 vs. 0.63±0.06, p-Akt protein (p-Akt/Akt): 0.98±0.10 vs. 0.75±0.07, all P < 0.05].

Conclusions: TFL regulates the expression of miR-146a to inhibit the PD-1/PD-L1 signaling pathway in THP-1 cells, regulates the immune barrier of sepsis induced in cell inflammation model in vitro, and thus protects LPS induced THP-1 cells.