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