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

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

2. The Hospital of Anhui University of Chinese Medicine, Hefei 230031, China

Keywords:Adjuvant arthritis miR145-5p/Smads pathways Macrophage polarization

ABSTRACT Objective: To explore the relationship between the changes of miR145-5p/Smads pathway and macrophage polarization in adjuvant arthritis rats. Methods: Twelve rats were divided into normal group and model group induced by freund's complete adjuvant (0.1 mL/mouse)by random number table method, with 6 rats in each group. The expression of inflammatory polarization markers IL-8 and CD206 in synovial tissue was detected by enzyme-linked immunosorbent assay on the 12th day after the formation of arthritis in rats. Western blotting was used to detect the expression of TGF-β1/Smads pathway factors in synovial tissues. The expression of miR145-5P, Smads3 and Smads7 in synovial tissue was detected by RT-qPCR.Results: Compared with normal group, the expression levels of IL-8, TGF-β1 and Smad3 in model group were significantly increased (P＜0.05); The expression levels of CD206, Smad7 and miR145-5P were significantly decreased (P＜0.01). The correlation results showed that IL-8 was positively correlated with Smad3 (P＜0.01), IL-8 was negatively correlated with Smad7 (P＜0.05), CD206 was negatively correlated with Smad3 (P＜0.01) and positively correlated with Smad7 (P＜0.05). miR145-5p was negatively correlated with Smad3 (P＜0.01)and positively correlated with Smad7 (P＜0.01). Conclusion: miR145-5p may inhibit the overactivation of TGF-β1/Smads pathway, regulate macrophage polarization, and inhibit the development of adjuvant arthritis by inhibiting Smad3 expression.

1. Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of synovial joints, pannus formation,progressive bone erosion, and joint destruction [1]. Related epidemiology shows that the incidence of this disease in China is 0.42%, with a high disability rate, which seriously affects the physical and mental quality of life of patients [2]. The pathogenesis of RA is still unclear, but a large number of studies have shown that it is related to joint synovitis caused by immune response[3].Macrophages are common immune cells that participate in human immune response by secreting and phagocytosing inflammatory mediators. Previous studies have shown that macrophages are involved in the development of RA disease[4]. Under the induction of different environments and factors, macrophages can successfully differentiate into M1-type macrophages with pro-inflammatory properties and M2-type macrophages with anti-inflammatory properties, which is the phenomenon of macrophage polarization,and the imbalance of polarization between macrophages may be an important feature of RA disease development [5-6]. However,the mechanism of macrophage polarization imbalance in RA disease development is still unclear [7]. Studies have found that macrophage polarization of RA may be related to the disorder of microRNA(miRNA) regulation and abnormal activation of TGFβ1/Smads signaling pathway [8-9]. TGF-β1/Smads can act on macrophages and immune cells through recruitment of inflammatory factors and cell proliferation. As a target, RA can be promoted.MiRNA is a highly conserved small non-coding RNA, and its role in a variety of diseases has been confirmed. The involvement of TGF-β1/Smads pathway in RA macrophage polarization may be associated with miR145-5p [10]. It has been reported that miR145-5p plays an important role in the development of RA disease and can regulate the proliferation and inflammatory response of RA fibroblast synovial cells, which may be a potential target for the treatment of RA [11-12]. Abnormal activation of miR145-5p/Smads signals may influence the inflammatory progression of RA, but its mechanism of action on macrophage polarization is unclear. Based on the adjuvantarthritis (AA) rat model, The correlation between miR145-5p/Smads signaling pathway and macrophage polarization in AA rats was investigated by observing the correlation between miR145-5p/Smads signaling pathway and the expression of major markers of M1 and M2 macrophages IL-8 and CD206.

2. Materials and methods

2.1 Material

2.1.1 Experimental animalsTwelve clean-grade MALE SD rats with body weight of (180±10)g were provided by Experimental Animal Center of Anhui Medical University. The animal license number was Lscxk (Anhui) 2017-001. The experiment was carried out after one week of adaptive feeding. The experiment was approved by the Experimental Animal Ethics Committee of our hospital (Ethics Number: AHUCM-RATS-2021022).

2.1.2 Main reagentsIL8 and CD206 were purchased from Wuhan Genomei Technology Co., LTD., the article numbers are JYM0583Ra and JYM1324Ra respectively. ECL kit was purchased from American Therm Company, the article number is 340958. Freund complete adjuvant(CFA) was purchased from Sigma Company, USA.

2.2 Methods

2.2.1 Animal grouping and modelingTwelve clean male SD rats were divided into normal group and model group by random number table method, with 6 rats in each group. CFA 0.1 mL was injected into the plantar skin of the right hind foot of each rat in the model group for modeling, and the AA rat model was made.

2.2.2 Enzyme Linked Immunosorbent assayEnzyme Linked Immunosorbent assay (ELISA) was used to detect the expression of macrophage polarization markers IL-8 and CD206 in synovial tissue of rat knee joint. The samples to be tested and blank Wells were set according to kit instructions, and incubated at 37 ℃ for 30 min. The absorbance of IL-8 and CD206 was measured with a microplate reader at 37 ℃ for 10 min, and the content of IL-8 and CD206 was calculated according to the standard curve of the specimen.

2.2.3 Western BlottingWestern Blotting (WB) was used to detect the expression of TGFβ1/Smads signaling pathway related factors in the synovial tissue of the knee of rats. The synovial tissue was lysed, the supernatant protein was collected after centrifugation, and the protein loading buffer was added for denaturation. Sds-page was applied with sample well electrophoresis. The proteins were transferred with PVDF membrane and then rinsed and sealed with 5% skim milk powder at room temperature for 2 h. Goat anti-rabbit IgG (1:1000)was incubated at 4 ℃ overnight and goat anti-mouse IgG (1:20 000)was incubated at room temperature for 1.2 h. After washing the membrane, ECL luminescence kit was used to detect the protein.Image J software was used to analyze the gray value of the target protein, and the relative expression levels of TGF-β1, Smad3 and Smad7 were calculated, and compared with the internal reference protein.

2.2.4 RT-qPCRRT-qPCR was used to detect miR145-5p, Smad3 and Smad7 gene expressions in synovial tissue of knee joint. Primers were shown in Table 1. Trizol method was used to extract synovial macrophage RNA, and PCR instrument was heated at 42 ℃ for 2 min and ice bath for 1min. Add the reaction solution and place it at 37 ℃ for 15 min. At 85 ℃ for 5 s, the reaction solution (cDNA) was removed as a template for fluorescence quantification. PCR amplification conditions: pre-denaturation at 95 ℃ for 1 min(one cycle),denaturation at 95 ℃ for 20 s, denaturation at 60 ℃ for 1 min, 40 cycles. Ct values of reference genes and target genes were collected by PCR, and β-actin was used as internal reference. Relative expression levels of miR-145-5p, Smad3 and Smad7 were calculated by 2-△△Ct method.

Table 1 Sequences of miR-145-5p receptors and ligand primers

2.2.5 Statistical processing

Count data were measured by mean ± standard deviation, and Spearman Spearman test was used for correlation analysis. P＜0.05 indicated statistically significant differences, and SPSS23.0 statistical software was used for analysis.

3. Results

3.1 Changes of markers of macrophage polarization in synovial tissue of AA rats

Compared with the normal group, IL-8 expression was significantly increased and CD206 expression was significantly decreased in the synovial membrane of the model group, and macrophages in the model group were polarized to M1-type, with statistically significant differences (P＜0.01), as shown in Table 2.

Table 2 Comparison of markers of M1 and M2 between the two groups(pg/mL,n=6,±s)

Table 2 Comparison of markers of M1 and M2 between the two groups(pg/mL,n=6,±s)

Group IL-8 CD206 Normal group 31.29±17.99 3 013.12±324.64 Model group 261.00±56.25 693.60±49.65 t-9.527 17.300 P＜0.001 ＜0.001

3.2 Changes of TGF-β1/Smads pathway factors in synovial tissues of AA rats

Compared with the normal group, the expression of TGF-β1 and Smad3 in the model group was increased, while the expression of Smad7 was significantly decreased, with statistical significance(P＜0.05), as shown in Table 3 and Figure 3 and Figure 1.

Table 3 Comparison of changes of TGF-β1/Smads pathway in synovium(n=6,±s)

Table 3 Comparison of changes of TGF-β1/Smads pathway in synovium(n=6,±s)

Group Smad3 Smad7 TGF-β1 Normal group 0.34±0.04 0.60±0.03 0.28±0.06 Model group 0.56±0.08 0.16±0.01 0.57±0.02 t-8.346 22.309 -4.101 P 0.015 ＜0.001 0.001

Figure 1 Changes of TGF-β1/Smads pathway in the two groups

3.3 miR145-5p /Smads pathway gene expression in synovial tissues of AA rats

Compared with the normal group, the expression of Smad3 was increased in the model group, while the expression of miR145-5p and Smad7 was decreased, with statistically significant differences(P＜0.01), as shown in Table 4 and Figure 4.

Table 4 Comparison of changes in miR145-5p /Smads pathway genes in synovium(n=6,±s)

Table 4 Comparison of changes in miR145-5p /Smads pathway genes in synovium(n=6,±s)

Group Smad3 Smad7 miR145-5p Normal group 1.01±0.12 1.00±0.43 1.00±0.07 Model group 1.68±0.09 0.74±0.06 0.61±0.04 t-13.57 10.38 14.49 P＜0.001 ＜0.001 ＜0.001

3.4 Analysis of macrophage polarization markers and correlation between miR-145-5p and TGF-β1/Smads pathway factors in AA rats

Spearman correlation analysis showed that M1 macrophage marker IL-8 was positively correlated with Smad3 (P＜0.01) and Smad7 (P＜0.05). M2 macrophage marker CD206 was negatively correlated with Smad3 (P＜0.01) and positively correlated with Smad7 (P＜0.05), miR145-5p was negatively correlated with Smad3(P＜0.01) and positively correlated with Smad7 (P＜0.01), and the differences were statistically significant. The above results indicate that the imbalance of macrophage M1/M2 is related to Smad3 and Smad7, and the expressions of Smad3 and Smad7 are regulated by miR145-5p, as shown in Table 5.

Table 5 Analysis of macrophage polarization markers and correlation between miR145-5p and TGF-β1/Smads pathway factors

4. Discussion

RA is an autoimmune mediated chronic joint inflammation accompanied by bone erosion and destruction, often involving damage to multiple organ systems [13]. At present, modern medicine cannot completely cure the disease, and long-term drug treatment is usually accompanied by serious side effects [14]. Therefore, the key to studying the progress of RA is to clarify the pathogenesis of articular cartilage destruction and synovial inflammation, and to find new targets to control the development of RA.

Studies have shown that macrophage polarization plays a key role in the progression of RA disease [15]. Polarized M1-type macrophages recruit inflammatory cells to produce inflammatory mediators, including TNF-α, IL-1, IL-8, etc., which trigger a series of inflammatory reactions, thus exacerbating joint inflammation.Polarized M2 macrophages can secrete anti-inflammatory cytokines,such as Arg1, IL-10, VEGF, CD206, etc., to promote tissue repair and immune regulation. To eliminate the inflammatory response.The polarization degree of macrophages in the synovial membrane of RA patients reflects the disease degree of RA. M1 macrophages are expressed in RA patients with high activity, while M2-type macrophages are mainly expressed in RA patients with low activity[16]. Macrophages polarize into M1-type differentiation, and the proinflammatory cytokines secreted by the cells, such as TNF-α and IL-6, not only aggravate the existing inflammatory response, but also aggravate bone erosion and joint injury by inducing osteoclast differentiation, inhibiting cell proliferation and inducing osteoblast apoptosis at the later stage of differentiation [17].

The pathogenesis of RA is related to the over-activation of TGFβ1/Smads signaling pathway [18]. Transforming growth factor-β1(TGF-β1) has the ability of tissue repair and regulation of immune response, and will accumulate in a large number of inflammatory areas in the early stage of RA. Under its chemotactic action, T cells will be rapidly recruited to the inflammatory site, resulting in massive proliferation of inflammatory cells and joint enlargement,aggravating synovial inflammation and accelerating the progression of RA [19-20]. TGF-β1 function activates downstream signals by binding to transmembrane receptor kinases [21-22], whose main downstream signals are the Smad family (SMAD1-SMAD8). The Transforming Growth factorβ receptor I (TβRI) can be activated by TGF-β1 and phosphorylates Smad3 protein. The phosphorylated Smad3 protein binds to Smad4 and translocates into the nucleus to induce transcription of multiple target genes. Smad7 can induce TβRI degradation, thereby blocking Smad3 phosphorylation,inhibiting TGF-β1 transmission, preventing inflammation from sustained damage to joints, and delaying the progression of RA disease. Macrophages play recognition and effect roles in RA immune inflammatory response [23]. Previous studies have shown that over-activation of TGF-β1/Smads signaling pathway leads to imbalance of M1/ M2-type macrophages, and macrophages differentiate into M1 type, secrete more pro-inflammatory factors and inflammatory mediators, participate in RA inflammatory response, and aggravate bone destruction [24]. Studies have shown that the involvement of TGF-β1/Smads pathway in RA macrophage polarization may be related to miR-145-5p [12].

miRNA is a non-coding single-stranded RNA family with about 22 nucleotides, which exists in multiple tissues and organs of the human body [25]. MiRNA can both promote inflammation and limit inflammation through negative feedback loops, playing an important role in the process of RA disease. Abnormal expression of miR-145-5p in synovial membrane and peripheral blood of RA patients may inhibit the degree of synovial inflammation of RA by affecting the activation of relevant signal pathways and the secretion of inflammatory factors [26-28]. Huang Xunwu et al verified that the direct target gene of miR-145-5p is Smad3 and miR-145-5p inhibits the expression of Smad3 gene by using luciferase assay and other methods [29]. The correlation analysis between miR-145-5p and Smad3 showed that miR-145-5p was negatively correlated with Smad3 and positively correlated with Smad7, indicating that miR-145-5p may regulate TGF-β1/Smads signaling pathway by acting on Smad3. Correlation analysis showed that IL-8, a marker of macrophage polarization M1, was positively correlated with Smad3 and negatively correlated with Smad7. M2 marker CD206 was negatively correlated with Smad3 and positively correlated with Smad7. These results suggest that miR-145-5p regulates macrophage polarization and inhibits inflammatory responses in vivo by regulating TGF-β1/Smads signaling pathway.

In conclusion, miR-145-5p may regulate the expression of Smad3,inhibit the over-activation of TGF-β1/Smads pathway, regulate the m2-type polarization of macrophages, and participate in the development of RA disease.

Author's contribution:

Fan Wen-jie: Conducting experiments, analyzing data, writing papers; Chen Xi: Design experiments, and put forward guidance on the paper; Wan Lei, Fan Hai-xia, Liu Tian-yang, Li Ming, Liu Lei,Ge Yao: Participated in the experiment content and collected data;Wang Qing-qing, Fei Chen-chen, Zhou Qian: Specimen collection.