WANG Qing-qing, SHEN Xi, WAN Lei, FAN Hai-xia, LIU Tian-yang, LI Ming, LIU Lei, GE Yao, FAN Wen-jie, FEI Chen-chen, ZHOU Qian

1.Graduate Department of Anhui University of Traditional Chinese Medicine ,Hefei 230038,China

2. Department of Rheumatology the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine,Hefei 230031,China

Keywords:

ABSTRACT Objective: To investigate the effect of miR145-5P overexpression on the polarization imbalance of synovial macrophages in patients with rheumatoid arthritis (RA). Methods:Human mononuclear cells (THP-1) at logarithmic growth stage were induced into M1-type macrophages, and RA synovial fibroblasts M1-type macrophages were co-cultured into synovial macrophages. Synovial macrophages were divided into four groups :RA group(blank group), TGF-β1 group (model group) and miR145-5P overexpression group (TGF-β1+miR145-5P mimics group) and miR145-5P overexpression negative control group (TGF-β1+miR145-5P-mimics -NC group). The blank group did not receive any treatment, and the other three groups were induced by TGF-β1 in the medium for 48 h. Transfection miR145-5p mimic and miR145-5P-mimics-NC were added to co-culture medium, and IL-6, IL-6 and IL-6 of synovial macrophages were detected by ELISA.CD163 expression. Rt-qpcr was used to detect miR145-5p mRNA, TGF-β1mRNA, Smad3mRNA,Smad7mRNA expression level.The expression of TGF-β1/Smads pathway related proteins was detected by Western Blotting.Results: Compared with blank group, IL-6 level was up-regulated (P＜0.01) ,and CD163 level was down-regulated in model group(P＜0.05) , suggesting that TGF-β1 could induce intensified immune inflammatory response. Compared with the negative miR145-5P overexpression control group and model group, The expression of miR145-5P overexpression group molecule CD163 was significantly increased by ELISA(P＜0.01), and the expression of inflammatory factor IL-6 was decreased (P＜0.05). PCR showed that miR145-5P mRNA expression level was significantly increased in miR145-5P overexpression group,Smad3 mRNA and TGF-β1 mRNA were significantly decreased, and Smad7 mRNA was significantly increased (P＜0.01).WB method showed that the anti-inflammatory protein Smad7 was significantly increased,while TGF-β1 and Smad3 were significantly decreased (P＜0.01). Transwell chamber results confirmed that miR145-5P overexpression group significantly reduced macrophage invasion(P＜0.01). Correlation analysis showed that miR145-5P was negatively correlated with Smad3 and positively correlated with Smad7 (P＜0.01). Conclusion: miR145-5P may inhibit macrophage polarization in RA patients by targeting Smad3 protein, negatively regulating TGF-β1/Smads pathway, and alleviating immune inflammation.

1. Introduction

Rheumatoid arthritis (RA) is a chronic disease with synovial joint erosion as the main pathological basis, in which macrophage polarization almost runs through the whole process of RA development and prognosis, by inhibiting or eliminating proinflammatory The development of macrophages to restore the balance of macrophages will be an effective method for the treatment of RA [1]. The TGF-β1/Smads pathway has been shown to be associated with synovial hyperplasia in RA patients and is considered an important target for RA therapy [2].MicroRNA(miRNA, miRNA) is a non-coding small RNA that can bind to target genes and plays an important role in regulating the development,proliferation and apoptosis of micro-macrophages [3]. Abnormal expression of miR145-5p in the blood and synovium of RA patients can cause irreversible joint damage in RA patients [4]. Activated synovial macrophages play an important role in generating dysregulated conditions that promote chronic inflammation in rheumatoid arthritis [5]. Relevant studies have confirmed that miR145-5p may regulate the TGF-β1/Smads pathway through the expression of target genes, which affects the degree of synovial inflammation[6-7]. In this study, synovial macrophages were cultured in vitro, and the differences in the expression levels of miR145-5p were analyzed for the effect of TGF-β1/Smads pathway macrophage polarization, which provided new ideas for the clinical treatment of RA.

2. Materials and methods

2.1 Materials

Human AB serum was purchased from Shanghai Suer Biotechnology Co, Ltd; microplate reader model RT-6000 was purchased from Redu Company; DMEM medium was purchased from Gibco Company of the United States; electric heating incubator model DNP-9052BS-Ⅲ was purchased from Shanghai Sanfa; Human interleukin 6 (IL-6) model JYM0140Hu &, human sCD163 model JYM2022Hu & were purchased from Wuhan Genemei Technology Co, Ltd.; centrifuge model JW3021HR Anhui Jiawen Company; PrimeScript? RT Reagent Kit with gDNA Eraser (reverse transcription kit) was purchased from Thermo Fisher Scientific; primer synthesis sequences were purchased fromSangon BiotechCompany; Novostart SYBR qPCR SuperMix Plus (fluorescentdye) kit was purchased from Roche Company;fluorescence quantitative PCR instrument model PIKOREAL 96 was purchased from Thermo Scientific Company; Western primary antibody and secondary antibody removal solution were purchased from Beyotime Company; Transfection-related miR145-5p was purchased from Ambion Company; Smad3 protein was purchased from abcam Company;Smad7 protein was purchased from Bioworld;TGF-β1 was purchased from abcam; GAPDH was purchased from Zsbio.

2.2 Methods

2.2.1 Culture of macrophages and synovial fibroblasts

Take healthy adult AB serum, separate peripheral blood mononuclear cells (PBMC) by Ficoll density gradient centrifugation,wash with washing solution (PBS containing 2% FBS), suspend the cells and count them, and take the human mononuclear cells in logarithmic growth phase. (THP-1) was inoculated into the well plate, added phorbol ester, and cultured for 48 h, that is,macrophages. After 24 h in 2 mL of IFN-γRPMI1640 complete medium, M1 macrophages could be induced. RA synovial fibroblasts were co-cultured with M1-type macrophages in a transwell coculture chamber.

2.2.2 Cell culture, grouping

Synovial macrophages were co-cultured in DMEM complete medium, and the co-cultured cells were divided into 4 groups:RA group (blank group), TGF-β1 group (model group), TGFβ1+miR145 -5p mimics (miR145-5p overexpression group), TGFβ1+miRNA-145-5p mimics-NC (miR145-5p negative control group).

2.2.3 Cell transfection

The co-cultured cells were seeded in 6-well plates, pre-stimulated with LPS for 24 h, and washed twice with PBS. The blank group did not receive any treatment, and the model group, overexpression group, and negative controlgroup were treated with TGF-β1 for 48 h for induction (the final concentration was 10 ng/mL).The overexpression group and the negative control group were transfected with Lipofect-mineTM2000 transfection reagent after 10 ng/ml TGF-β1 treatment for 48 h. miR145-5p mimics and miRNA-145-5p mimics-NC were transfected into co-cultured cells, cultured for 6 h after transfection, and then replaced with low-glucose DMEM medium containing 10% AB serum for 24 h, and cells in each group were collected.

2.2.4 ELISA method to detect the expression of synovial macrophages

After 24 h of transfection and culture, the cell culture supernatant was taken and centrifuged at 1000 × g for 20 min, and the supernatant was taken. The concentration of IL-6 and CD163 was determined by enzyme-linked immunosorbent assay (ELISA), and the absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm.

2.2.5 qRT-PCR to detect the expression of miR145-5p and TGF-β1/Samd pathway

Take each group of cells and add Trizol reagent to extract total RNA. The ratio of A250/A270 is between 19 and 201 for reverse transcription, and PCR kit is used for amplification according to the reference [8]. miR145-5p used U6 as the internal reference, using the Relative Quantification Study, the calculation method was 2-△△Ct.The relative expression levels of miR145-5p, TGF-β1 mRNA and Smad3 mRNA, Smad7 mRNA were calculated. The sequences of the primers used in the experiments are shown in Table 1.2.2.6 Westernblot detection of Smad protein expression

Tab 1 Primer sequences

Collect cell samples (about 1×105cells) or tissue samples (about 0.1 g), and add 600 μL of RIPA cell lysis buffer (containing 0.6 mM PMSF) for lysis. Centrifuge at 12 000 r/min for 15 min. Referring to the reference method [9], SDS-PAGE electrophoresis, membrane transfer solution, blocking, primary antibody and secondary antibody dilution (primary antibody dilution ratio 1:1 000, secondary antibody dilution ratio 1:20 000), and room temperature incubation were carried out respectively. After adding washing solution (PBST)to wash the membrane, the color was developed to detect the protein.

2.2.7 Transwell cell migration assay

After culturing the Transwell chamber for 24 h (37℃), the cells on the microporous membrane that did not pass through the chamber membrane were wiped off; the cells were treated with 4%paraformaldehyde solution for 20 min at room temperature, poststained for 15 min, and observed under a light microscope at will.The number of cells passing through the membrane of the Transwell chamber in 3 fields.

2.2.8 CCK8 assay to detect cell growth

48h after transfection, trypsinized log-phase cells were inoculated into 96-well plates with 100 μL cell suspension per well, and blank control group and model group were set up with only culture medium but no cells inoculated, with 3 replicates in each group.hole. After 48 hours of incubation (37℃, 5% CO2), 10 μL of CCK8 was added to each well, and the culture was continued for 2 hours.The absorbance at 460 nm was measured with a microplate reader.

2.3 Statistical processing

Statistical software SPSS26.0 data analysis software and GraphPad Prism 7 drawing software were used to process data. Measurement data were expressed as±s, and Spearman analysis was used for correlation analysis, and P＜0.05 indicated that the difference was statistically significant. Pairwise comparisons within groups were performed using LSD-t test, and data comparisons between multiple groups were performed using ANOVA, with P＜0.05 being considered statistically significant.

3. Results

3.1 The effect of TGF-β1 induction on the expression level of synovial

Macrophages and the effect of miR145-5p overexpression on the expression of inflammatory factors in synovial macrophages The experimental results showed that compared with the blank group, the synovial MI macrophage IL-6 in the TGF-β1 group was significantly increased (P＜0.01), and the M2 macrophage molecule CD163 was significantly decreased (P＜0.05), suggesting that TGFβ1 -β1-induced stimulation can exacerbate inflammatory responses.Compared with the model group and miR145-5p overexpression negative control group, the level of anti-inflammatory molecule CD163 in synovial macrophages in the miR145-5p mimics group was significantly increased (P＜0.01), and the pro-inflammatory factor IL-6 was significantly lower (P＜0.01). ＜0.05), suggesting that overexpression of miR145-5p can inhibit the expression of pro-inflammatory factors and promote the polarization of M1-type macrophages to M2-type macrophages. See Table 2.

Tab 2 Effects of TGF-β1 on the expression of IL-6 and CD163 in synovial macrophages (n=6,±s)

Tab 2 Effects of TGF-β1 on the expression of IL-6 and CD163 in synovial macrophages (n=6,±s)

Note: Compared with blank group, ▲P＜0.05, ▲▲P＜0.01; compared with model group and overexpression negative control group, *P＜0.05, **P＜0.01.

Groups IL-6(ng/mL) CD163(pg/mL)RA 16.42±1.31 8387.08±1223.47 TGF-β1 TGF-β1+miR145-5p mimic TGF-β1+miR145-5p mimics- 109.15±17.56▲▲1342.53±231.01▲s 54.71±5.23* 4205.62±402.77▲▲**NC 106.31±16.38 8081.49±1766.91 F 78.496 56.201 P＜0.001 ＜0.05

3.2 Relationship between miR145-5p overexpression and TGF-β1/Smads pathway gene expression

PCR results showed that, compared with the model group and the miR145-5p overexpression negative control group, the miR145-5p-mimics group could significantly up-regulate the expression level of miR145-5p in co-cultured cells and inhibit the expression of TGF-β1 mRNA in co-cultured cells. The level of Smad3 mRNA was significantly decreased, and Smad7 mRNA was significantly increased (P＜0.01), the difference was statistically significant,see Table 3. It is suggested that overexpression of miR145-5p can regulate the TGF-β1/Smads pathway by inhibiting the expression of TGF-β1 and Smad3, promoting the expression of Smad7.

3.3 The effect of miR145-5p overexpression on synovial macrophage-related proteins

The results of Western blot experiments showed that the expression levels of marker protein bands in the four groups were significantly different, as shown in Figure 1. The Smad7 protein in the miR145-5p overexpression group was significantly higher than that in the model group and the miR145-5p overexpression negative control group, and the difference was statistically significant (P＜0.01), as shown in Table 4. It is suggested that miR145-5p mimics can inhibit the expression of TGF-β1 and Smad3 protein and increase the expression of Smad7 protein in synovial macrophages.

Fig 1 Expression of TGF-β1/Smads signaling pathway related proteins in synovial macrophages of each group

Tab 3 Effects of miR145-5P overexpression on TGF-β 1-induced Smad protein gene (n=6,±s)

Tab 3 Effects of miR145-5P overexpression on TGF-β 1-induced Smad protein gene (n=6,±s)

Note: Compared with the model group and miR145-5p overexpression negativecontrol group, ▲▲P＜0.01, **P＜0.01.

Groups Smad3 Smad7 TGF-β1 miR145-5p RA 1.03±0.49 TGF-β1 2.43±1.45 TGF-β1+miR145-5p mimics 1.79±0.39▲TGF-β1+miR145-5p mimics-NC 2.32±0.141.00±0.23 0.99±1.06 1.02±0.60 0.49±0.46 3.07±0.26 0.61±0.80▲** 0.75±0.51▲▲** 2.20±0.74▲▲** 4.2±0.61▲▲**0.53±0.72 3.00±0.21 1.09±0.97 F 50.505 127.288 34.371 51.920 P＜0.001 ＜0.001 ＜0.001 ＜0.001

Tab 4 Expression of TGF-β1/Smads pathway proteins in synovial macrophages (n=6,±s)

Tab 4 Expression of TGF-β1/Smads pathway proteins in synovial macrophages (n=6,±s)

Note: Compared with the model group and miR145-5p overexpression negativecontrol group, ▲▲P＜0.01, **P＜0.01.

Groups Smad3 Smad7 TGF-β1 RA 0.17±0.09 TGF-β1 0.63±0.39 TGF-β1+miR145-5p mimics 0.20±0.50▲▲**TGF-β1+miR145-5p mimics-NC 0.58±0.45 F 154.770.86±0.11 0.32±0.02 0.18±0.98 0.67±0.10 0.60±0.52▲▲** 0.32±0.95▲▲** 0.48±0.29 0.55±0.11 40.31 29.75 P＜0.001 ＜0.001 ＜0.001

3.4 The effect of miR145-5p overexpression on the migration of RA synovial macrophages

The migration ability of cells was detected by CCK-8, observed under a transweill chamber microscope, and the migration experiment was visible.The transmembrane cells in the miR145-5p overexpression group were significantly less than those in the RA group, the model group and the overexpression negative control group (P＜0.01), as shown in Figures 2 and 3. The results of transwell chamber showed that overexpression of miR145-5p significantly reduced macrophage invasion and improved inflammatory response.

3.5 Correlation analysis of miR145-5p and M1, M2 macrophage markers and Smads protein expression

Spearman correlation analysis showed that miR145-5p was negatively correlated with Smad3 (P＜0.01), and positively correlated with Smad7 (P＜0.01); Smad7 was positively correlated (P＜0.05),and the correlation between M2 macrophage molecule CD163 and Smad3 and Smad7 was opposite to that of IL-6. The results of correlation analysis indicated that miR145-5p could regulate macrophage polarization by inhibiting Smad3 expression, as shown in Table 5.

Fig 2 Picture of transmembrane cells under microscope(×100)

Fig 3 Number of transmembrane cells in microscopic field

Tab 5 Correlation analysis of miR145-5P and macrophage markers and Smads protein expression

4. Discussion

Rheumatoid arthritis is a common clinical and progressive immune disease. There is increasing evidence that macrophage polarization plays a pivotal role in the development of RA disease [10].Macrophages are divided into two subgroups. After polarization, M1 macrophages secrete a large number of pro-inflammatory cytokines,chemokines, etc. to activate fibroblasts and osteoclasts, triggering a series of inflammatory responses, resulting in the destruction of articular cartilage, M2-type macrophages express antagonistic proinflammatory molecules on the surface to promote tissue repair and reduce inflammatory responses [11-12]. Relevant studies have found [13-14] that in the blood and synovial tissues of RA patients and mouse models, macrophages and M1 and M2 markers are unbalanced on the surface of cells, and the M1/M2 ratio in the synovium increases, promoting Osteoclast formation and enhanced immune response. Therefore, intervening in the transformation of macrophages from M1 type to M2 type can restore the dynamic balance of M1/M2, which is beneficial to promote the repair of RA synovial inflammation.

Transforming growth factor-β (TGF-β) is a multipotent growth factor that stimulates the secretion and deposition of extracellular matrix, and plays an important role in inducing apoptosis, bone formation, regulating hematopoiesis and immune responses and other rheumatic immune diseases. regulating effect. Sun Wenkui[15]and others found that the high expression of TGF-β1 in synovial macrophages of RA patients is specific, and the high expression of TGF-β1 in synovial macrophages can promote the expression of M1-type pro-inflammatory factors in RA patients. Thereby promoting its inflammatory reaction, leading to the recurrence and development of RA patients. Zhu DingJi[16] et al. confirmed that TGF-β1 was highly expressed in fibroblast-like synovial cells (FLSs) and synovial fluid of RA patients, and TGF-β1 enhanced the migration and invasion of RA. In this experiment, synovial macrophages were induced and stimulated by TGF-β1, and synovial macrophages without TGF-β1 induction and stimulation were used as control. The results of ELISA showed that M1 macrophages in the model group secreted IL-6.When the level increased, the secretion level of M2-type macrophage molecule CD163 decreased, indicating that TGF-β1 stimulation can aggravate the inflammatory response of synovial macrophages,which is also consistent with the previous related research results.

TGF-β1 can act on macrophages and immune cells by recruiting inflammatory factors, cell proliferation, etc., and activate the downstream Smads protein family. target [17]. SMAD protein is an important downstream pathway of TGF-β intracellular signaling.After phosphorylation by TGF-β receptor-I (TβRI), it forms an isoform complex with the receptor-regulated protein SMAD3,phosphorylates and activates downstream SMAD proteins. Smad3 induces transition to mesenchymal-upperplasm and promotes RA pathology. Smad3 gene polymorphisms may lead to perturbation of the TGF/Smad signaling pathway, which affects T cell responses and gene expression through multiple mechanisms [18], which may lead to inhibition of trans growth factor (TGF)-β-mediated induction of Treg cells, and induction of Th1 and Th17 development. Smad7 is an inhibitory protein, and Smad7 can cause the degradation of TβRI, thereby blocking the phosphorylation of Smad3, inhibiting the conduction of TGF-β1, preventing the continuous damage caused by inflammation to the joints, and delaying the progression of RA disease. Smad7 deletion may stimulate Th cell differentiation and autoantibody production, thereby promoting the progression of autoimmune arthritis, enhancing NF-κB activation, Th1/Th17 differentiation and synovial inflammation [19]. Therefore, reducing Smad3 protein expression and increasing Smad7 protein expression may be the key steps to delay synovial inflammation in RA joints.

miRNAs can regulate gene expression post-transcriptionally and then regulate the expression of downstream Smad proteins through complementary pairing with target genes, and play an important role in the mechanism of RA development. As one of the miRNA family members, miR145-5p has been studied more deeply in recent years on the effect of miR145-5p expression on RA. Ming Jiansong [20]and others successfully established a collagen-induced mouse model and found that miR145-5p inhibited joint inflammation in CIA mice. Qiu JianLi [21] et al. isolated and knocked out recombinant viruses by lentivirus-mediated overexpression of miR145 and found that miR145 enhanced the TGF-β/Smad pathway, leading to the activation of stellate cells. The PCR results of this study showed that compared with the other three groups, the miR145-5p mRNA expression was significantly increased, the Smad3 mRNA expression was significantly decreased, and the Smad7 mRNA expression was significantly increased in the miR145-5p overexpression group. ELISA results showed that miR145-5p overexpression group inhibited the polarization of M2-type macrophages to M1-type macrophages, decreased the expression of M1-type pro-inflammatory factors, and increased the expression of M2-type anti-inflammatory factors; transwell chamber results showed that miR145-5p was overexpressed Significantly reduced macrophage invasion. All the above evidences suggest that miR145-5p overexpression can regulate macrophage polarization balance.

Based on ROC analysis, Paradowska et al. [22] believed that Smad3 may be an important biomarker for clinical experiments and diagnosis of rheumatoid arthritis. Yu C et al. [23] used dual-luciferase reporter gene analysis, Smad3, a key element of the TGF-β1 inflammatory pathway, was the target of miR145. Yu Fang-Yuan[24]confirmed that Smad3 is the direct target gene of miR145 binding to 3'-UTR by using the online prediction method. Overexpression of miR145 significantly inhibited the expression of Smad3 mRNA and protein, and partially inhibited osteoclast differentiation. In this experiment, WB was used to detect TGF-β1,The expression levels of Smads pathway-related proteins were analyzed, and the results showed that Smad3 was decreased, Smad7 was increased,the expression of M1-type pro-inflammatory factors was decreased,and the expression of M2-type anti-inflammatory factors was increased in the miR145-5p overexpression group. At the same time, correlation analysis showed that miR145-5p was negatively correlated with Smad3, and the correlation between M1 and M2 macrophage polarization markers and Smad3 and Smad7 was also confirmed by PCR and WB results.

In summary, the following conclusions were drawn in this study:miR145-5p overexpression may regulate Smad3 protein through negative feedback, inhibit the expression of inflammatory factors in the TGF-β1/Smads pathway, and inhibit the transition of M2 macrophages to M1 type, thereby maintaining macrophages. The dynamic balance of phagocyte polarization may be a new target for clinical experiments and treatment of RA.

Author Contribution Description:

Wang Qingqing: conducting cell experiments, recording and analyzing data, and writing papers; Chen Xi: designing experiments and providing guidance on the papers; Wan Lei, Fan Haixia,Liu Tianyang, Li Ming, Liu Lei, and Ge Yao: participating in experimental content and collecting data; model essay Jie, Fei Chenchen, Zhou Qian: Specimen collection.