Paeoniflorin reduces the inflammatory response of THP-1 cells by up‐regulating microRNA-124

Background The activation of macrophages and the release of inflammatory cytokines are the main reasons for the progress of systemic lupus erythematosus (SLE). MicroRNA (miRNA)-124 is involved in the regulation of macrophages and is a key regulator of inflammation and immunity. Objective To explore whether paeoniflorin (PF) regulates the biological functions of macrophages depends on miR-124. Methods RT-PCR, WB, ELISA, CCK-8 and flow cytometry were used to evaluate that PF regulated the biological functions of THP-1 cells through miR-124. Results PF significantly inhibited the proliferation while promotes the apoptosis of THP-1 cells, and inhibited the release of IL-6, TNF-α and IL-1βin THP-1 cells. RT-PCR results shown that PF up-regulated the expression of miR-124 in THP-1 cells. Functional recovery experiments showed that compared with the LPS + mimic-NC group, LPS + miR-124 mimic significantly inhibited the proliferation and the release of IL-6, TNF-α and IL-1β, but promoted the apoptosis of THP-1 cells. In addition, compared with the LPS + PF + inhibitor-NC group, LPS + PF + miR-124 inhibitor significantly promoted the proliferation and the release of IL-6, TNF-α and IL-1β, but inhibited the apoptosis of THP-1 cells. Conclusions By down-regulating miR-124, PF inhibits the proliferation and inflammation of THP-1 cells, and promotes the apoptosis of THP-1 cells.


Introduction
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease, and innate immune cells and cytokines play key roles in its pathology (Kiriakidou and Ching 2020). Studies on SLE patients and animal models have shown that the activation of macrophages and the abnormalities of related cytokines are closely related to SLE (Ma et al. 2019;Zhang et al. 2020). Macrophages are mainly divided into two subtypes, M1 (pro-inflammatory phenotype) and M2 (antiinflammatory phenotype) (Funes et al. 2018). And induction of macrophage polarization into the M2 phenotype is helpful to reduce experimental SLE . Therefore, the inhibition of macrophage proliferation and induction of M2 macrophage polarization are helpful to alleviate the disease progression of SLE.

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Recently, studies have reported that paeoniflorin (PF), an extract of paeonia lactiflora root, inhibits the proliferation of a variety of cells, such as human mesangial cells (Liu et al. 2019), B cells (Zhang et al. 2015) and vascular smooth muscle cells (Fan et al. 2018). However, the effect of PF on the proliferation and the polarization of macrophages are not yet clear. And, we speculate that PF might exert certain clinical value in the treatment of SLE patients via regulating the proliferation and the polarization of macrophages.
MicroRNA (miRNA)-124 has become a key regulator of inflammation and immunity, and is involved in the regulation of various innate and adaptive immune cells, including hematopoietic cells, CD4 + T cells and macrophages (Qin et al. 2016). Among them, miR-124 inhibits the proliferation of macrophages (Zhai et al. 2020), and is essential for inducing and maintaining the M2 phenotype of macrophages (Veremeyko et al. 2013). Therefore, we speculate that PF might inhibit the proliferation and promote the polarization of macrophages by promoting the expression of miRNA-124.
Based on the basis above, this study aims to explore whether there exists the potential mechanism of PF/miRNA-124 in macrophages, and the effects of PF/miRNA-124 on the proliferation and polarization of macrophages. These findings of this study might provide new ideas for the treatment of SLE patients.

Cell viability assays
Cell viability was measured by using a Cell Counting Kit-8 (CCK-8, Dojindo, Japan) according to the manufacturer's instructions. Briefly, cells were seeded at a density of 0.3 × 10 4 cells/well in 96-well plates, and incubated in 1640 medium overnight. Then, CCK-8 solution (10 ul) and 90 ul 1640 medium (serum free) was added to each well for 2 h and optical density was measured at 450 nm. The proliferation of cells was detected at 12 h, 24 h, 36 and 24 h.

Western blot
We lysed the cells using a protein extraction reagent (Beyotime, Jiangsu, China) in the presence of protease and phosphatase inhibitor cocktail tablets. We measured protein concentration using a BCA Protein Assay Kit (Beyotime, Jiangsu, China). Soluble lysates containing about 50 µg proteins per sample were resolved with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Merck Millipore). After blocking using 10 % fat-free milk, membranes were incubated with primary antibodies against IL-6 (1:100, CST), TNF-α (1:100,CST), IL-1β (ab234437, abacam) and β-actin (1:100, CST) overnight at 4 °C overnight and secondary antibodies (1:1000) at room temperature for 1 h. After several washes, the immunoblot was detected with enhanced chemi-luminescence (Pierce Biotechnology) according to the manufacturer's instructions.

Apoptosis assay
After drug treatment for 48 h, cells were harvested and washed twice with PBS and resuspended in binding buffer, then the cells were double-stained with Annexin V-Phycoerythrin (PE) and 7-aminoactinomycin (7-AAD) (BD Biosciences) for 15 min at room temperature in the dark. Subsequently, cells were analyzed by flow cytometry using Calibur (BD Biosciences) within 1 h.

ELISA
The Human TNF alpha ELISA Kit (ab181421, abacam), Human IL-6 ELISA Kit (ab178013, abacam) and Human IL-1 beta ELISA Kit (ab214025, abacam) were used to detected the expression of TNF-α, IL-6 and IL-1β in the cell lysate and cell supernatant of THP-1 cells. Each experiment was repeated at least three times.

Statistical analysis
The SPSS version 21.0 software was used for data analyses. Statistical significance was confirmed using a Student's t test. Statistically significant differences were defined as p < 0.05.

PF inhibits the cell function and inflammation of THP-1 cells induced by LPS
The molecular formula of PF (PubChem ID: 442,534) is C 23 H 28 O 11 , with molecular weight: 480.5 g/mol. As provided by PubChem database, the 2D and 3D structure of PF were shown in Fig. 1a and b. Through CCK-8 detection, we found that 500-3000 ug/ml PF significantly inhibited the cell viability of THP-1 cells (Fig. 1c). Since M2 type macrophages help to improve SLE , 1000-3000 ug/ml PF seems to be over-dose for macrophages. Therefore, we choose 500 ug/ml PF for follow-up experiments. Through ELISA experiments, we found that 500 ug/ml PF inhibited the expression of TNF-α, IL-6 and IL-1βin the cell lysate (Fig. 1d) and cell supernatant (Fig. 1e) of THP-1 cells. These results suggest that 500ug/mL PF can not only substantially inhibit the proliferation of THP-1 cells, but also substantially reduce the secretion of TNF-α, IL-6 and IL-1βin THP-1 cells. It is suggested that 500 ug/mL PF may reduce the secretion of TNF-α, IL-6 and IL-1β by inhibiting the proliferation of THP-1 cells.
Through bioinformatics prediction via the TCMSP database, we found that PF might be involved in the regulation of TNF-α, IL-6, CD14 and LPS-binding protein (Fig. 2a). CCK-8 and flow cytometry detection showed that 500 ug/ ml PF could significantly inhibit the cell viability (Fig. 2b) while promote the apoptosis (Fig. 2c) of THP-1 cells. In addition, the increase in mRNA (Fig. 2d) and protein ( Fig. 2e-g) expression of TNF-α, IL-6 and IL-1β induced by LPS could be significantly reversed by 500 ug/ml PF. Together, 500ug/ml PF could inhibit the pro-inflammatory response induced by LPS.

LPS regulates the cell function and inflammation of THP-1 cells by inhibiting the expression of miR-124
Through the bioinformatics prediction via the miRwalk, Targetscan and miRDB database, we found that there are 14 potential miRNA targets for LPS-binding protein (Fig. 3a). Therefore, we speculate that LPS might be involved in regulating the expression of these miRNAs. RT-PCR results showed that LPS inhibited the expression of miR-124.
However, 500 ug/ml PF significantly reversed the downregulation of miR-124 induced by LPS (Fig. 3b). RT-PCR results showed that the expression of miR-124 in the miR-124-mimic group was significantly increased by about 6 times compared with the mimic-NC group (Fig. 4a). And overexpression of miR-124 significantly inhibited the increase in cell viability induced by LPS by CCK-8 detection (Fig. 4b). The results of flow cytometry showed that overexpression of miR-124 significantly promoted the apoptosis of THP-1 cells (Fig. 4c). In addition, overexpression of miR-124 could significantly reverse the increase of TNF-α, IL-6 and IL-1β expression in mRNA (Fig. 4d) and protein ( Fig. 4e-g) levels induced by LPS. Together, PF regulates the biological function and inflammation of THP-1 cells by down-regulating the expression of miR-124.

ug/ml PF up-regulates miR-124 to regulate the biological functions induced by LPS in THP-1 cells
As shown in Fig. 3a, PF significantly promoted the expression of miR-124 in THP-1 cells. RT-PCR results showed that compared with the inhibitor-NC group, the expression of miR-124 was significantly reduced in the miR-124-inhibitor group (Fig. 5a). CCK-8 results showed that compared with the PF + LPS + inhibitor-NC group, the cell viability of THP-1 cells in the PF + LPS + miR-124-inhibitor group was significantly increased (Fig. 5b). The results of flow cytometry showed that compared with the PF + LPS + inhibitor-NC group, in the PF + LPS + miR-124-inhibitor group, the apoptosis of THP-1 cells was significantly reduced (Fig. 5c). In addition, compared with the PF + LPS + inhibitor-NC group, the mRNA (Fig. 5d) and protein ( Fig. 5e-g) expression of TNF-α, IL-6 and IL-1β in the PF + LPS + miR-124-inhibitor group increased significantly. Together, 500ug/ml PF inhibits the activity and inflammation of THP-1 cells by upregulating the expression of miR-124.

Discussion
In this work, we characterized the biological role of PF in THP-1 cells, a macrophage suppressor that inhibits cell proliferation and promotes cell apoptosis by up-regulating the expression of miR-124. In addition, PF inhibits the expression of TNF-α, IL-6 and IL-1β by up-regulating miR-124. PF has been found to inhibit tumor growth in a variety of tumors (Zhang et al. 2018). However, whether PF inhibits the growth of macrophages remained unknown. In the present study, we found for the first time that 500 ug/ml PF could significantly inhibit the proliferation of THP-1 cells, but 1, 10 and 100 ug/ml PF have no significant effect on the biological function of THP-1 cells. In addition, M2 type macrophages is beneficial for improving SLE ) and thus 1000-3000 ug/ml PF seems to be over-dose for macrophages. Therefore, 500 ug/ml PF were used in the subsequent experiments to further explore the biological function of PF in THP-1 cells.
LPS has the function of promoting cell proliferation and inflammation (Fritsche 2015). In this study, we found that LPS promotes the proliferation and the apoptosis of THP-1 cells. In addition, LPS can significantly promote the release of IL-6, IL-1β and TNF-α in THP-1 cells. However, 500ug/ ml PF significantly reversed the pro-proliferation and proinflammatory effects induced by LPS in THP-1 cells. Therefore, PF may reduce the release of IL-6, IL-1β and TNF-α by inhibiting the proliferation of THP-1 cells.
IL-1β and TNF-α have strong pro-inflammatory activity and can promote the secretion of a variety of pro-inflammatory mediators, including IL-6, IL-8 and IL-l0 . Zhu et al. (2018)reported that compared with healthy controls, IL-6 was elevated in the plasma of leukemia patients, and IL-6 supported the survival of leukemia cells in vivo/in vitro. Parnsamut and Brimson (2015) reported that gold nanoparticles inhibited the cell proliferation of monocytic U937 cells by down-regulating the expression of TNF-α. These results suggest that the inhibition of IL-6, IL-1β and TNF-α by PF may further reduce the proliferation of THP-1 cells.
Abnormal expression of miR-124 has been found in many inflammatory and immune diseases, and it often acts as a negative regulator of inflammation or immune signals (Qin et al. 2016). In our study, 500ug/ml PF was found significantly up-regulated the expression of miR-124 in THP-1 cells. Further function gain-and-loss experiments indicated that the increase in cell viability and the release of IL-6, IL-1β and TNF-α induced by 100 ng/mL LPS were significantly reversed by the miR-124. As reported before, miR-124 inhibits the proliferation of macrophages (Zhai et al. 2020), and inhibits the expression of TNF-α, IL-6 and IL-1β (He et al. 2019;Ohnuma et al. 2019). In addition, miR-124 is essential for inducing and maintaining the M2 phenotype of macrophages (Veremeyko et al. 2013). Therefore, PF may inhibit the proliferation of THP-1 cells and the release of IL-6, IL-1β and TNF-α by up-regulating miR-124. However, whether PF directly inhibits the release of IL-6, IL-1β and TNF-α in THP-1 cells by up-regulating miR-124, in a way Fig. 1 PF inhibits the proliferation and the release of pro-inflammatory cytokines of THP-1 cells. The 2D (a) and 3D (b) structures of PF. c 500 ug/ml PF significantly inhibited the proliferation of THP-1 cells. ELISA results showed that PF inhibited the protein expression of IL-6, IL-1β and TNF-α in the cell lysate (d) and supernatant (e) of THP-1 cells. *Represents p < 0.05, NS represents no significant difference ◂ that does not depend on the inhibition of proliferation, needs further verification.
In conclusion, this study initially elucidates that PF inhibits the proliferation and the release of proinflammatory cytokines of THP-1 cells by promoting the expression of miR-124 (Fig. 6). And, our preliminary results support the clinical potential of PF for the treatment of SLE patients.  Compared with the LPS + mimic-NC group, the proliferation of THP-1 cells in the LPS + miR-124-mimic group decreased significantly (b), while the apoptosis increased significantly (c). Compared with the LPS + mimic-NC group, the mRNA (d) and protein (e-g) expression of IL-6, IL-1β and TNF-α in the LPS + miR-124-mimic group were significantly reduced via RT-PCR, WB and ELISA detection. *Represents p < 0.05; U6, β-actin and GAPDH are used as an internal control Fig. 5 500 ug/ml PF significantly reversed the cell function and inflammation induced by LPS in THP-1 cells. a The efficiency of miR-124-inhibitor was tested by RT-PCR. Compared with the PF + LPS + inhibitor-NC group, the proliferation of THP-1 cells in the PF + LPS + miR-124-inhibitor group increased significantly (b), while the apoptosis decreased significantly (c). Compared with the PF + LPS + inhibitor-NC group, the mRNA (d) and protein (e-g) expression of IL-6, IL-1β and TNF-α in the PF + LPS + miR-124-inhibitor group were significantly increased via RT-PCR, WB and ELISA detection. *Represents p < 0.05; U6, β-actin and GAPDH are used as an internal control