Effect of Xifeng Capsule on blood stasis in patients with rheumatoid arthritis by regulating miR-126-VEGF/PI3K/AKT signaling pathway

Zhang-Ying Lin, Yuan Wang, Chuan-Bing Huang, Wan-Dong Zhang, Gui-Zhen Wang,Rui-Lian Chen, Xue Gong

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

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

ABSTRACT Objective: From the perspective of miR-126-vascular endothelial cytokine (VEGF)/phosphatidylinositol 3-kinase (PI3K)/ser-threonine protein kinase (AKT) signaling pathway,Xinfeng Capsule (XFC) can improve rheumatoid arthritis (RA) ) The mechanism of the patient's blood stasis state. Methods: Sixty RA patients meeting the diagnostic criteria were selected and divided into XFC treatment group 30 cases and Leflunomide (LEF) control group 30 cases according to the random number table method. The treatment group took Xinfeng Capsules (3 capsules each time, 3 times/d), and the control group took Leflunomide(1 capsule each time, 1 times/d). Observe the blood stasis symptom scores of RA patients,and detect the laboratory indicators erythrocyte sedimentation rate (ESR), c-reactive protein(CRP), rheumatoid factor (RF), anti-cyclic citrullinated peptide antibody (CCP), thrombin time (TT), part prothrombin time (APTT), prothrombin time (PT), D dimer (DD), fibrinogen(FBG), platelets (PLT), mean platelet volume (MPV), platelet distribution width (PDW)levels. Real-time fluorescent quantitative PCR method was used to detect the level of miR-126, and the ELISA method was used to detect the levels of tumor necrosis factor (TNF-α),interleukin-6 (IL-6), IL-35, VEGF, PI3K, and AKT. Spearman method was used to analyze the correlation between the total score of blood stasis symptoms, blood stasis-related indicators and disease activity indicators, cytokines, miR-126, VEGF, PI3K, and AKT in RA patients. Results: Comparing the two groups after treatment, the XFC group improved blood stasis symptoms, disease activity indicators, decreased miR-126, VEGF, PI3K, AKT,TNF-α, IL-6, D-D, FBG, PLT, and increased IL-35 The level was significantly better than the LEF group, with statistical significance (P<0.05, P<0.01). Correlation analysis showed that there was a certain correlation between the total score of blood stasis symptoms, blood stasis related indicators and disease activity indicators, cytokines, miR-126, VEGF, PI3K,and AKT in RA patients. Conclusion: There is blood stasis in RA patients. XFC may improve the cytokine network disorder of patients through miR-126-VEGF/PI3K/AKT signaling pathway, thereby improving the blood stasis status of RA patients.

Keywords:Rheumatoid arthritis Blood stasis state Xinfeng capsule miR-126 VEGF/PI3K/AKT pathway?Corresponding author: WANG Yuan, Chief Physician, Associate Professor, M.D..E-mail: echowang0268@126.com.

1. Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized by target organ damage due to chronic inflammation of the synovial membrane of the joints and immune system dysfunction,which can seriously affect the quality of life of patients. Mainly manifested as destruction of bone and articular cartilage, vasculitis and blood stasis, etc[1-2]. The basic pathological changes of RA include the formation of synovitis, pannus and vasculitis, which are closely related to the hypercoagulable state, vascular endothelial hyperplasia, and abnormal elevation of platelets in the patient's body. RA belongs to the category of "Wang Bi". Traditional Chinese medicine believes that long-term illness must result in blood stasis.Blood stasis is an important cause of lingering and difficult healing of the disease. Blood stasis is the main cause and pathological product of RA. At the same time, its main pathogenesis runs through the occurrence and development of RA disease. The research team believes that spleen deficiency and dampness, qi and blood deficiency, and mutual accumulation of phlegm and blood stasis are the basic pathogenesis of RA. Put forward the treatment method of "Yiqi, tonify the spleen and dredge collaterals", create Xinfeng Capsule (XFC) for clinical application, and the curative effect is remarkable. Previous studies have found that hypercoagulable state is common in RA patients[2]. XFC may increase the level of the anti-inflammatory factor interleukine-10 (IL-10) and reduce the level of the inflammatory factor IL-17 and IL-6 by inhibiting the abnormally activated NF-κB pathway, thereby regulating Cytokine network. So that the partial prothromboplastin time (activated partial thromboplastin time, APTT), prothrombin time (PT),thrombin time (TT), D dimer (d-dimer, DD), fibrinogen (FBG),platelets (platelet, PLT), platelet activating factor, platelet activating factor-acetylhydrolase tend to a new equilibrium state, thereby improving the blood stasis state of RA patients [3]. At the same time,we found in the clinic that the expression of vascular endothelia growth factor (VEGF) in RA patients increased, and it was closely related to disease activity [4]. VEGF is an extremely important proangiogenic factor that can bind to receptors on the synovial vascular endothelium, promote synovial angiogenesis and pannus formation,increase vascular permeability and the release of inflammatory factors [5], thereby participate in the pathological process of RA blood stasis. The phosphoinositide 3-kinases (PI3K)/serine-threonine kinase (AKT) signaling pathway is related to the mechanism of microvascular damage and angiogenesis [6], which is VEGF An extremely important part of the downstream signaling pathway,which can play a regulatory role in angiogenesis. Overexpression of PI3K or AKT can induce extensive new angiogenesis and enlarge existing blood vessels [7], which is closely related to the blood stasis state of RA patients. MiR-126 is a positive regulator in the angiogenesis signaling pathway. Overexpression of miR-126 can activate the VEGF/PI3K/AKT signaling pathway, enhance vascular permeability, and promote the formation of synovial pannus [8].In this paper, by observing the changes of miR-126-VEGF/PI3K/AKT signaling pathway, explore the possible mechanism of XFC to improve RA blood stasis.

2. Materials and methods

2.1 Research object

Sixty patients with RA who were hospitalized in the Department of Rheumatology and Immunology of our hospital were selected,and the hospital stay was from May 2020 to December 2020.According to the random number table, 30 cases were divided into XFC treatment group and 30 cases in leflunomide (LEF) control group. There was no statistically significant difference between the two groups of data (P>0.05), and they were comparable.This study has been approved by the Ethics Committee of Anhui Provincial Hospital of Traditional Chinese Medicine, and patients need to sign an informed consent form.

2.2 Diagnostic criteria

According to the RA classification standard jointly proposed by the American College of Rheumatology (ACR) and the European Union Against Rheumatism (EULAR) in 2010 [9], RA can be diagnosed with a total score of ≥6.

2.3 The scoring standard of blood stasis syndrome

According to the "Guiding Principles for Clinical Research of New Chinese Medicines", the standard of TCM syndromes formulated the "Symptom Grading and Quantitative Table of Blood Stasis Syndrome" [10]. According to different degrees of joint tingling, lip color, tongue quality, pulse condition, subcutaneous ecchymosis,and skin nail fault, they are counted as normal, mild, moderate, and severe, respectively, as 0, 1, 2, and 3 points.

2.4 Inclusion criteria

①M(fèi)eet the above diagnostic criteria; ② Meet the diagnostic criteria for the active phase; ③ Age 18 to 75 years; ④ Patients who have used non-steroidal anti-inflammatory drugs and slow-acting anti-rheumatic drugs must be stopped for more than 3 months before admission Group. ⑤ Sign the informed consent form voluntarily.

2.5 Exclusion criteria

①Patients who do not meet the above diagnostic criteria;②Combined with serious diseases and severe extra-articular manifestations; ③Patients with mental disorders; ④Pregnant or breastfeeding women; ⑤Those who are considered unsuitable for this clinical trial by the investigator.

2.6 Method

2.6.1 Treatment plan

The treatment group was given Xinfeng Capsules (3 tablets, 3 times/d), and the control group was given Leflunomide tablets (1 tablets, 1 time/d) for 30 consecutive days. In the study, the two groups of patients can use non-steroidal anti-inflammatory analgesics according to the situation, but avoid the use of immunosuppressive agents and other proprietary Chinese medicines for the treatment of RA.2.6.2 Laboratory index testing

DD, FBG, TT, APTT, PT, PLT, mean platelet volume (MPV),platelet distribution width (PDW), erythrocyte sedimentation rate(ESR), C-reactive protein (c -reactive protein (CRP), rheumatoid factor (rheumatoid factor, RF), and anti-cyclic citrullinated peptide(CCP) were all tested by Anhui Provincial Hospital of Traditional Chinese Medicine.

2.6.3 ELISA to detect serum

According to the ELISA kit instructions, the levels of VEGF, PI3K,AKT, tumor necrosis factor-α (TNF-α), IL-6 and IL-35 were determined.

2.6.4 Detection of miR-126 by real-time fluorescence quantitative PCR

The content of miR-126 was detected according to the instructions of the real-time fluorescent quantitative PCR kit.

2.6.5 Statistical methods

SPSS 21.0 software was used for data analysis, and the data were expressed as ±s. The two groups were compared by t test or nonparametric test, and the correlation analysis was performed by spearman analysis with a test level of 0.05.

3. Results

3.1 Comparison of blood stasis symptom scores between the two groups before and after treatment

Compared with before treatment, the blood stasis symptoms and signs of the two groups were significantly improved after treatment(P<0.05, P<0.01). Compared with the LEF group, the joint tingling,tongue quality, subcutaneous ecchymosis, and blood stasis total scores were significantly improved in the XFC group after treatment,with statistical differences (P<0.05). See Table 1.

3.2 Comparison of inflammatory indexes and cytokines between the two groups before and after treatment

Compared with before treatment, CRP, ESR, RF, CCP, IL-6,TNF-α and other indicators of the two groups were significantly decreased after treatment, and IL-35 was significantly increased, and the difference was statistically significant (P<0.01) . Compared with the LEF group, IL-35 in the XFC group was significantly higher than that in the LEF group after treatment, and ESR, TNF-α, and IL-6 were significantly reduced, and the difference was statistically significant (P<0.05, P<0.01). See Table 2.

3.3 The influence of the two groups on blood stasis indicators in RA patients

Compared with before treatment, the DD, FBG, and PLT of the two groups decreased after treatment, the difference was statistically significant (P<0.01), APTT, TT, PT, MPV, PDW did not change significantly, and the difference was not statistically significant(P> 0.05). Compared with the LEF group, the D-D, FBG, and PLT decreased more significantly in the XFC group, and the difference was statistically significant (P<0.01). See Table 3.

Table1 Comparison of blood stasis symptom score between two groups before and after treatment compare(±s )

Table1 Comparison of blood stasis symptom score between two groups before and after treatment compare(±s )

Note: Compared with this group before treatment, *P<0.05, **P<0.01; compared with the same period in the LEF group, △P<0.05, △△P<0.01. (The same as Table 2-4)

Index XFC group (n=30) LEFgroup(n=30)Before treatment After treatment Before treatment After treatment Blood stasis total score 10.83±1.74 6.67±1.27**△ 10.7±1.86 9.30±1.58**Joint tingling 1.83±0.65 0.50±0.51**△ 1.80±0.55 1.20±0.61**Lip color 2.07±0.64 1.90±0.55* 2.03±0.49 1.97±0.49*Tongue quality 2.13±0.68 0.67±0.66**△ 1.73±0.69 1.83±0.75**Pulse condition 1.70±0.65 1.63±0.61 2.10±0.66 1.60±0.62*Subcutaneous ecchymosis 1.47±0.78 0.53±0.68**△ 1.43±0.82 1.23±0.63*Squamous and dry skin 1.63±0.49 1.43±0.63* 1.60±0.56 1.47±0.51*

Table 2 Comparison of inflammatory indicators and cytokines between the two groups before and after treatment(±s)

Table 2 Comparison of inflammatory indicators and cytokines between the two groups before and after treatment(±s)

Index XFC group(n=30) LEF group(n=30)Before treatment After treatment Before treatment After treatment CRP(mg/L) 17.91±14.18 7.30±5.82** 17.19±9.69 9.05±6.57**ESR(mm/h) 36.8±18.71 10.97±5.15**△ 36.30±16.13 13.93±5.04**RF(U/ml) 127.88±99.17 90.37±66.78** 127.91±90.81 89.66±58.61**CCP(U/ml) 171.52±203.54 135.78±156.66** 172.16±162.10 144.31±142.20**IL-6(pg/ml) 64.87±3.70 46.30±12.20**△△ 65.57±4.18 56.62±9.05**TNF-α(pg/ml) 402.20±66.69 293.70±76.07**△ 401.23±65.13 333.50±79.09**IL-35(pg/ml) 34.32±3.97 42.45±7.14**△△ 35.88±3.11 38.23±3.65**

3.4 The effects of the two groups on miR-126, VEGF, PI3K,and AKT in RA patients

Compared with before treatment, the levels of miR-126, VEGF,PI3K, and AKT were significantly reduced in the two groups after treatment, and the difference was statistically significant (P<0.01).Compared with the LEF group, the levels of VEGF, miR-126, PI3K,and AKT in the XFC group decreased more significantly after treatment, and the difference was statistically significant (P<0.05,P<0.01). See Table 4.

3.5 Correlation analysis of blood stasis indicators and cytokines, miR-126, VEGF, PI3K, AKT in RA patients

The blood stasis total score of RA patients is positively correlated with CRP, ESR, RF, CCP, TNF-α, IL-6, miR-126, VEGF, PI3K,AKT, and negatively correlated with IL-35 expression (P<0.05, P<0.01). D-D is positively correlated with CRP, ESR, CCP, IL-6, and VEGF (P<0.05, P<0.01). FBG was positively correlated with CRP,CCP, VEGF, PI3K, and AKT (P<0.05, P<0.01). PLT was positively correlated with CRP, ESR, RF, CCP, TNF-α, IL-6, VEGF, PI3K, and AKT (P<0.01). MPV is negatively correlated with PI3K (P<0.01);PDW is negatively correlated with CCP (P<0.05). See Table 5.

4. Discussion

RA belongs to the categories of "calendar day disease","rheumatism", "crane knee wind" and "white tiger disease" in Traditional Chinese medicine. It was seen early in "Suwen·Bilun",and it is related to the three qi of wind, cold and dampness.The onset of RA is not only related to external pathogenic factors such as wind,cold and dampness, but also related to internal causes."Yi Zong Jin Jian·Summary of Arthralgia" pointed out: "Spleen deficiency means that people with deficiency of qi are diseased with all kinds of arthritis." Point out that deficiency of spleen qi and lack of righteousness are the main pathological basis of RA blood stasis.In clinical practice, the team applied "treatment from the spleen" to the clinic, and developed Xinfeng Capsules for the treatment of RA.Xinfeng Capsules are mainly composed of four drugs: Astragalus,Coix Seed, Tripterygium, and Centipede.Liu Yun et al. [11] found that early intervention of Astragalus injection can regulate the levels of thrombin-antithrombin complex and platelets, and improve microcirculation disorders. Coix seed sprout extract can inactivate the PI3K/AKT pathway, resulting in prolonged cell cycle and apoptosis, and reduced angiogenesis [12]. At the same time, coix seed injection can down-regulate the expression of vascular endothelial cytokines in experimental abdominal adhesion rats, thereby further inhibiting the release of fibrinogen, and at the same time reducing the expression levels of pro-inflammatory cytokines IL-1 and TNF-α[13], It has a regulatory effect on the blood stasis state of RA.Triptolide can inhibit the pathological aggregation of platelets, and at the same time it also has the effect of regulating the body's blood stasis state [14]. Centipede can significantly prolong the TT, APTT, and PT of mice [15] and improve the blood stasis state. Throughout the whole prescription, XFC has the effects of invigorating the spleen,dispelling dampness, promoting blood circulation and clearing collaterals, and has obvious curative effect on RA blood stasis state.At this stage, the pathogenesis of blood stasis syndrome has not been fully studied, but most researchers believe that its specific manifestations are hyperviscosity and hypercoagulability, and blood coagulation and platelet-related indicators are important laboratory indicators for judging blood stasis syndrome[16-18]. Therefore, blood coagulation and platelet-related indicators can help to detect the changes in the blood stasis state of patients in the early stage of RA disease to guide the clinical selection of prescriptions and drugs,and better delay the disease progression of RA patients. Our team’s previous study found that [19], compared with the normal group,DD, FBG, and PLT in the peripheral blood of RA patients were significantly increased (P<0.01), and TT was prolonged (P<0.01).The coagulation in the peripheral blood of RA patients was related to platelets. The indicators are positively correlated with clinical symptoms and signs, DAS28, and disease activity indicators.Therefore, the prevention and improvement of blood stasis status in RA patients have important clinical significance. This study found that the XFC group can improve disease activity indicators, blood stasis symptoms and signs, and reduce the total score of blood stasis symptoms, DD, FBG, and PLT (P<0.05, P<0.01). Compared with the LEF group, the XFC group is treated The post-decrease was more obvious (P<0.05, P<0.01), suggesting that Xinfeng Capsule can improve the blood stasis state while alleviating the activities of RA patients.

Table 3 Effects of two groups on blood stasis related indexes in RA patients(±s )

Table 3 Effects of two groups on blood stasis related indexes in RA patients(±s )

Index XFC group(n=30) LEF group(n=30)Before treatment After treatment Before treatment After treatment D-D(mg/L) 1.35±1.04 0.43±0.37**△△ 1.36±1.01 0.66±0.42**FBG(g/L) 4.38±1.09 2.69±0.61**△△ 4.35±1.01 3.03±0.57**TT(sec) 16.08±0.75 16.35±0.85 16.01±0.75 16.38±0.96 APTT(sec) 26.34±1.32 26.81±1.57 26.22±1.42 27.86±1.60 PT(sec) 10.02±0.50 10.00±0.53 10.05±0.54 10.05±0.52 PLT(109/L) 248.73±56.56 180.87±45.60**△△ 246.73±56.22 210.10±43.55**MPV(fL) 10.98±1.44 10.89±1.17 10.84±1.20 10.79±1.02 PDW(fL) 12.55±2.63 12.83±2.25 12.64±1.93 13.33±2.41

Table 4 Effects of the two groups on miR-126, VEGF, PI3K and AKT in RA patients (±s )

Table 4 Effects of the two groups on miR-126, VEGF, PI3K and AKT in RA patients (±s )

Index XFC group(n=30) LEF group(n=30)Before treatment After treatment Before treatment After treatment VEGF(pg/ml) 398.70±121.34 81.13±27.31**△△ 399.17±197.56 97.83±33.98**PI3(pg/ml) 2989.62±630.09 800.95±393.58**△ 2901.14±640.15 1300.46±390.69**AKT(pg/ml) 2452.68±366.97 1068.47±236.56**△ 2440.59±750.46 1392.56±342.92**miR-126 16.58±0.88 11.18±1.95**△ 16.56±0.96 14.08±1.88**

Table 5 Correlation analysis of blood stasis indicators with cytokines, miR-126, VEGF, PI3K and AKT(r)

The mechanism of blood stasis in RA may be closely related to angiogenesis and the formation of pannus. VEGF is an inducing mediator of angiogenesis [20] and a key factor in the formation of pannus. At the same time, VEGF can act as a specific mitogen of vascular endothelial cells in the signal-level angiogenesis of downstream PI3K-Akt, promote endothelial cell proliferation and migration, and can also promote the secretion of interstitial collagenase and degrade cell matrix [21]. PI3K/Akt, as a downstream signaling pathway of VEGF, can promote endothelial cell proliferation and angiogenesis through PI3K-Akt downstream molecule mammalian target of rapamycin complex 2 and forkhead transcription factor [22-23], and participate in the state of blood stasis.miR-126 can negatively regulate the expression of VEGF to regulate vascular endothelial cells [24] and promote angiogenesis, which is closely related to the state of RA blood stasis. At the same time, miR-126 can regulate the PI3K/AKT signaling pathway by affecting the PIK3R2 gene to exert its functions in angiogenesis, cell proliferation,differentiation and migration [25]. Studies have pointed out that in in vitro experiments, IL-35 can inhibit the migration of human umbilical vein endothelial cells, tube formation and the secretion of Angiopoietin 2 induced by VEGF [26]. TNF-α can promote the adhesion of endothelial cells to leukocytes, thereby stimulating endothelial cells to secrete inflammatory substances, thereby activating the coagulation system and inhibiting fibrinolysis [27],and the binding of TNF to its receptors can affect the proliferation and migration of VEGF and affect rheumatoid joints. The level of vascular proliferation in patients with inflammation leads to bone and cartilage damage [28]. IL-6 promotes RA angiogenesis mainly through the synergistic effect of TNF-α, IL-1 and IL-17 to increase the expression of VEGF and CCL28 [29]. This study found that the blood stasis total score and blood stasis-related indicators of RA patients are correlated with TNF-α, IL-6, IL-35, miR-126, VEGF,PI3K, and AKT. It shows that the blood stasis state of RA patients is closely related to the miR-126-VEGF/PI3K/AKT signaling pathway and inflammatory factors. After XFC treatment, the levels of disease activity indicators, miR-126, VEGF, PI3K, AKT, TNF-α, IL-6, DD,FBG, PLT decreased significantly (P<0.01), and IL-35 increased significantly (P< 0.01), suggesting that XFC may have an inhibitory effect on the miR-126-VEGF/PI3K/AKT signaling pathway, downregulating the levels of pro-inflammatory factors IL-6 and TNF-α,and up-regulating the levels of anti-inflammatory factors IL-35,thereby reducing DD, FBG, PLT level, alleviate RA blood stasis state.

In summary, the abnormal activation of miR-126-VEGF/PI3K/AKT signaling pathway is closely related to the blood stasis state of RA patients. XFC can improve the blood stasis state of RA patients.The mechanism may be through inhibition of miR-126-VEGF The activation of the /PI3K/AKT signaling pathway regulates the cytokine network, thereby delaying or inhibiting blood stasis and reducing joint disease. In this study, only clinical studies have been carried out, and further cell experiments will be carried out to verify in future experiments to improve the reliability of the conclusions.

Author’s contribution

First author: Lin Zhangying Completed data collection, data analysis, and paper writing;

Corresponding author: Wang Yuan designed the experiment and provided guidance on the paper;

Other authors: Huang Chuanbing, Zhang Wandong, Wang Guizhen,Chen Ruilian, Gong Xue participated in research design, research implementation and data collection.