Gout is caused by monosodium urate monohydrate (MSU) depositions in synovial fluid and other tissues and characterized by bursal or peripheral joint swelling, tenderness, and pain, with MSU crystals in a symptomatic joint, bursa or tophus [
Mice with a miR-146a deficiency develop severe gouty arthritis as a result of upregulation of the expression of TRAK6, IRAK-1 and NALP3 inflammasome, thus increasing the degree of the inflammatory response [
The aims of the present study were to screen differentially expressed miRNAs in CGA patients and healthy controls using miRNA microarray assay technology and to verify differentially expressed miRNAs via RT-qPCR. At the same time, we studied the effects of the Chuanhutongfeng mixture in the treatment of CGA and on differentially expressed miRNAs, which might have a regulatory role in CGA.
From May 2014 to February 2015, 255 patients with CGA in gout clinics of the Affiliated Hospital of Qingdao University and 30 age and gender-matched healthy subjects were selected for the trial. The fasting plasma of all participants was collected. The study protocol was approved by the Research Ethics Committee of the Affiliated Hospital of Qingdao University, and all patients provided written informed consent.
The expression of miRNAs in plasma from eight subjects was screened by miRNA chip technology. There were four patients with CGA (3 males, 1 female: 50.20±8.90 years) and four healthy controls (3 males, 1 female: 49.70±8.70 years). There was no significant difference in age and gender between the 2 groups (Figure
Flowchart of the study. CCL2: chemokine (C-C motif) ligand 2; CXCL8: chemokine (C-X-C motif) ligand 8; CRP: C-reactive protein; ESR: erythrocyte sedimentation rate.
After obtaining expression profiles of plasma miRNAs, RT-qPCR was used to validate further the differentially expressed miRNAs in the CGA (60 patients) and control (30 healthy subjects) groups. There were 60 patients with CGA including 56 males and 4 females (50.80±9.00 years) and 30 healthy controls including 28 males and 2 females (50.00±8.90 years). There was no significant difference in age and gender between the two groups (Table
Baseline data for high-throughput gene screening and RT-qPCR validation in the healthy control and CGA groups.
Indicators | Healthy control group (n = 30) | CGA group | |
---|---|---|---|
Age | 50.00 ± 8.90 | 50.80 ± 9.00 | 0.691 |
Gender (% of males) | 28 (93.33) | 56 (93.33) | 1.000 |
BMI (kg/m2) | 25.20 ± 1.10 | 25.50 ± 1.30 | 0.281 |
Systolic pressure (mmHg) | 132.00 ± 8.00 | 133.00 ± 7.00 | 0.544 |
Diastolic pressure (mmHg) | 76.00 ± 6.00 | 77.00 ± 6.00 | 0.458 |
FBG (mmol/L) | 5.10 ± 0.60 | 5.20 ± 0.50 | 0.406 |
TC (mmol/L) | 4.90 ± 0.50 | 5.10 ± 0.50 | 0.077 |
TG (mmol/L) | 2.10 ± 0.80 | 2.20 ± 0.90 | 0.608 |
ALT (U/L) | 30.00 ± 9.00 | 31.10 ± 8.50 | 0.572 |
AST (U/L) | 25.20 ± 7.80 | 26.80 ± 8.30 | 0.382 |
eGFR(ml/min/1.73m2) | 111.00 ± 6.50 | 110.00 ± 7.50 | 0.392 |
SUA ( | 330.00 ± 70.00 | 529.00 ± 76.00 | < 0.001 |
ESR (mm/h) | 10.00 ± 5.50 | 33.00 ± 15.70 | < 0.001 |
CRP (mg/L) | 3.10 ± 2.20 | 22.20 ± 11.70 | < 0.001 |
Pain scores | 0 | 3.60 ± 0.80 | - |
Swelling scores | 0 | 1.90 ± 0.89 | - |
Restricted movement scores | 0 | 1.90 ± 0.90 | - |
CGA: chronic gouty arthritis; BMI: body mass index; FBG: Fasting blood-glucose; TC: total cholesterol; TG: triglyceride; ALT: alanine aminotransferase; AST: aspartate aminotransferase; eGFR: estimate glomerular filtration rate; SUA: serum uric acid; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein.
Total RNA of plasma samples was isolated using Trizol LS (Invitrogen, Canada) and purified with an RNeasy mini kit (Qiagen, Germany), and the quantity of RNA was determined using a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, US). A miRCURY™ Array Power Labeling kit (Exiqon, Denmark) was used for microRNA labeling, and then Hy3™-labeled samples were hybridized on a miRCURY™ LNA Array (Exiqon). Chips were scanned using an Axon GenePix 4000B microarray scanner (Axon Instruments, CA, US) and the images were imported into GenePix Pro 6.0 software (Axon Instruments, CA, US) for grid alignment and data extraction. The replicated microRNAs were averaged and those with intensities ≥ 30 in all samples were selected for normalization. Differentially expressed microRNAs were identified by a volcano plot and a heat map (MEV ver. 4.8, TIGR).
To establish the accuracy of the microRNA assays, we used RT-qPCR to validate differentially expressed miRNAs in the plasma samples. Total RNA was isolated using Trizol LS (Invitrogen, Canada) and purified with an RNeasy mini kit (Qiagen, Germany), and the quantity of RNA was determined using a NanoDrop 8000 spectrophotometer (
The design was a 1:1:1 prospective, randomized, double blind, double simulation, and parallel controlled study. It was estimated that ≥ 50 cases would be needed for each group, the estimated rate of abscission was about 20%, and 195 cases were included. Nomenclature has been used according to previous literature [
First, we selected the appropriate segment length and then determined the number of seeds according to Proc Plan in SAS statistical software, resulting in a random arrangement of 195 patients in the group.
The principle of double blinding was carried out including the generation of random numbers, the number of experimental drugs, the specific grouping of research subjects, the recording, management, and statistical analysis of data.
Chuanhutongfeng mixture is an improvement of the Tongfeng mixture which we developed before and contains
The control group was given 30 mg etoricoxib orally, once a day, a placebo tablet of allopurinol, and a placebo agent of the Chuanhutongfeng mixture for a total course of treatment of 8 weeks. The Chuanhutongfeng mixture group was based on the control group plus Chuanhutongfeng mixture (hospital preparation, unified decoction) orally 2 times a day and a placebo tablet of allopurinol for a total course of treatment of 8 weeks. The allopurinol group was based on the control group plus allopurinol 100 mg administered orally 3 times a day and a placebo agent of the Chuanhutongfeng mixture.
Height, weight, blood pressure, and related biochemical indicators were measured before 4 and 8 weeks after treatment for all subjects. FPG (after overnight fasting for 10 h) was measured using the glucose oxidase method; serum ALT, AST, creatinine, serum uric acid, and blood lipids, including TG and TC, were measured using an automated biochemical analyzer (7600-020; Hitachi, Tokyo, Japan). Serum CRP levels were determined using a particle-enhanced immunoturbidimetric assay (Beckman Coulter Inc., Kraemer Blvd., CA, US).
The curative effects including joint pain, swelling, and restricted movement scores were used in a range of scores, with a higher score reflecting the severity of the condition according to the following criteria after 4 weeks and 8 weeks of treatment.
The score was assessed by a numerical rating scale (NRS): no pain: 0 points; mild pain, tolerable, being able to live and sleep normally: 1-3 points; moderate pain, appropriate to affect sleep but tolerable: 4-6 points; severe pain with persistent pain, affecting sleep, requiring painkillers, or being associated with other symptoms or compulsive position: 7-10 points.
There is no significant change in skin texture and apophysis, without joint effusion: 0 points; skin texture becoming lighter, and the apophysis being clearly visible with a small amount of joint effusion: 1 point; the skin texture basically disappearing, the apophysis being flat with the swollen skin, and the signs of the apophysis being not obvious with medium joint effusion: 2 points; the skin texture completely disappearing, the swelling skin being higher than the apophysis, the signs of apophysis disappearing, and the joint function and activity being limited with a mass joint effusion: 3 points.
Joint movement was not restricted: 0 points; the movement was slightly restricted and could be engaged in normal activities: 1 point; restricted movement was obvious and not being able to engage in normal activities but being able to still take care of themselves: 2 points; the movements were completely limited, and patients could not take care of themselves on a daily basis: 3 points.
According to the related CGA efficacy criteria utilized by Meng [
This included the following: the correlation between clinical effects of CGA and expression levels of miR-339-5p, miR-486-5p, and miR-361-5p in the plasma of CGA patients before and after treatment.
This included the following: the levels of CCL2, CXCL8, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) in the plasma before and after the treatment.
The intention-to-treat (ITT) analysis and per-protocol treated (PP) analysis were used in AE analyses. All cases lost in the Chuanhutongfeng mixture, allopurinol, and control groups were considered to be acute CGA attacks.
SPSS ver. 16.0 statistical software (SPSS Inc., Chicago, IL, US) was used for data analysis. Normally distributed data are expressed as the
There were no significant differences in the baseline data and biochemical indexes compared to the healthy group, but SUA, ESR, and CRP were significantly elevated and there were gouty arthritis symptoms such as pain, swelling, and restricted movement scores in the CGA group (Table
Heatmap (Supplementary Figure
There were 48 miRNAs with higher or lower expression levels in the plasma of patients with CGA, of which 36 were significantly upregulated ≥ 1.5 times compared to the control group. miRNAs which were upregulated or downregulated > 2 times are shown in Supplementary Table
Based on GO analysis and pathway analysis, the pathways of the target genes that may be regulated by differentially expressed microRNAs were analyzed (Supplementary Figure
According to the literature and our analysis of expression differences, we chose three miRNAs that may play important roles in the pathogenesis of CGA (miR-339-5p, miR-486-5p, and miR-361-5p). RT-qPCR was used to detect microRNA expression in plasma samples obtained from 60 patients with CGA and 30 healthy controls. The relative expression levels of miR-339-5p, miR-486-5p, and miR-361-5p in the CGA group of patients were significantly higher than in the healthy control group (4.28-fold, 7.02-fold, and 5.83-fold, respectively) (Table
The relative expression of 3 miRNAs in plasma in the CGA and healthy control groups.
Healthy control group | CGA group | | |
---|---|---|---|
miR-486-5p | 17.12 ± 2.33 | 2.44 ± 1.26 | < 0.001 |
miR-339-5p | 20.44 ± 2.89 | 4.79 ± 2.13 | < 0.001 |
miR-361-5p | 18.18 ± 3.23 | 3.12 ± 1.44 | < 0.001 |
CGA: chronic gouty arthritis.
From May 2014 to February 2015, there were 255 cases of CGA in the gout clinic of the Affiliated Hospital of Qingdao University, and finally 195 cases were enrolled. There were 186 males and 9 females, with an average age of 50.50±9.30 years. In the allopurinol group, there were a total of 55 cases comprised of 53 males and 2 females, with an average age of 50.50±8.90 years. In the Chuanhutongfeng mixture group, there were 58 patients in the Chuanhutongfeng mixture group comprised of 55 males and 3 females, with an average age of (51.10±9.10) years. Finally there were 52 patients in the control group, comprised of 50 males and 2 females with an average age of 49.90±9.00 years.
All patients have been in treatments before the trial. There were no statistical differences in age, gender, miR-339-5p, miR-486-5p, miR-361-5p, blood pressure, FPG, blood lipids, serum uric acid, body mass index, eGFR, ALT, AST, CCL2, CXCL8, CRP, pain scores, swelling scores, and restricted movement scores between the three groups (Table
Baseline information of patients in the 3 groups.
Indicators | Control group | Allopurinol group | Chuanhutongfeng mixture group (n = 58) | |
---|---|---|---|---|
Age | 49.90 ± 9.00 | 50.50 ± 8.90 | 51.10 ± 9.10 | 0.784 |
Gender (% of males) | 50 (96.15) | 53 (96.36) | 55 (94.83) | 0.908 |
BMI (kg/m2) | 25.33 ± 1.12 | 25.46 ± 1.06 | 25.50 ± 1.26 | 0.726 |
Systolic pressure (mmHg) | 133.00 ± 7.00 | 132.00 ± 8.00 | 134.00 ± 7.00 | 0.354 |
Diastolic pressure (mmHg) | 76.00 ± 5.00 | 75.00 ± 7.00 | 75.00 ± 6.00 | 0.618 |
Disease duration (years) | 9.90 ± 5.11 | 9.91 ± 5.42 | 9.87 ± 5.50 | 0.999 |
Tophus (n, %) | 26 (50.00) | 28 (50.91) | 30 (51.72) | 0.984 |
FBG (mmol/L) | 5.12 ± 0.58 | 5.21 ± 0.46 | 5.17 ± 0.63 | 0.710 |
TC (mmol/L) | 4.89 ±0.45 | 5.03 ± 0.46 | 5.10 ± 0.47 | 0.057 |
TG (mmol/L) | 2.17 ± 0.85 | 2.21 ± 0.72 | 2.16 ± 0.81 | 0.940 |
ALT (U/L) | 30.20 ± 9.50 | 29.50 ± 8.80 | 31.70 ± 8.40 | 0.406 |
AST (U/L) | 25.10 ± 7.70 | 24.80 ± 7.40 | 27.90 ± 8.50 | 0.074 |
eGFR(ml/min/1.73m2) | 110.10 ± 7.40 | 111.50 ± 7.50 | 110.40 ± 7.70 | 0.339 |
SUA ( | 532.00 ± 69.00 | 529.00 ± 72.00 | 530.00 ± 74.00 | 0.976 |
ESR (mm/h) | 31.00 ± 14.20 | 33.90 ± 15.70 | 32.20 ± 16.30 | 0.622 |
CRP (mg/L) | 21.30 ± 9.20 | 20.40 ± 10.80 | 22.00 ± 12.10 | 0.734 |
Pain score | 3.54 ± 1.57 | 3.71 ±1.58 | 3.64 ±1.38 | 0.843 |
Swelling score | 1.97 ± 0.89 | 1.97 ± 0.95 | 1.97 ± 0.88 | 1.000 |
Restricted movement score | 1.98 ± 0.72 | 1.98 ± 0.89 | 1.99± 0.88 | 0.997 |
CCL2 (pg/mL) | 538.00 ± 223.00 | 545.00 ± 175.00 | 572.00 ± 160.00 | 0.597 |
CXCL8 (pg/mL) | 619.00 ± 199.00 | 643.00 ± 181.00 | 645.00 ± 218.00 | 0.757 |
miR-486-5p | 2.51 ± 1.22 | 2.31 ± 1.19 | 2.24 ± 1.31 | 0.505 |
miR-339-5p | 4.90 ± 1.98 | 4.93 ± 2.16 | 4.64 ± 2.18 | 0.725 |
miR-361-5p | 3.27 ± 1.80 | 3.15 ± 1.77 | 2.98 ± 1.47 | 0.661 |
BMI: body mass index; FBG: Fasting blood-glucose; TC: Total cholesterol; TG: triglyceride; ALT: alanine aminotransferase; AST: aspartate aminotransferase; eGFR: estimate glomerular filtration rate; SUA: serum uric acid; ESR: erythrocyte sedimentation rate; CRP: C-reactive protein; CCL2: chemokine (C-C motif) ligand 2; CXCL8: chemokine (C-X-C motif) ligand 8.
Improvement in the clinical symptoms of the three groups of patients after 4/8 weeks of treatment is shown in Table
Comparison of clinical efficacy in the 3 groups after 4 and 8 weeks treatment.
Clinical efficacy index | Control group | Allopurinol group | Chuanhutongfeng mixture group | |||
---|---|---|---|---|---|---|
4 weeks | 8 weeks | 4 weeks | 8 weeks | 4 weeks | 8 weeks | |
Cure | 3 (5.77%) | 4 (7.69%) | 18 (32.73%) | 27 (49.09%) | 22 (37.93%) | 42 (72.41%) |
Markedly effective | 10 (19.23%) | 12 (23.08%) | 20 (36.36%) | 15 (27.27%) | 20 (34.48%) | 12 (20.69%) |
Effective | 27 (51.92%) | 25 (48.08%) | 15 (27.27%) | 11 (20.00%) | 15 (25.86%) | 4 (6.90%) |
Ineffective | 12 (23.08%) | 11 (21.15%) | 2 (3.64%) | 2 (3.64%) | 1 (1.72%) | 0 (0.00%) |
SUA values were significantly more reduced after 4 weeks and 8 weeks of treatment in the Chuanhutongfeng mixture and the allopurinol groups compared to the control, but the SUA reduction in the Chuanhutongfeng mixture patients was significantly greater than in the allopurinol group after 8 weeks of treatment.
Comparison of differences in observation indicators after 4 and 8 weeks treatment in the 3 groups.
Indicators | Control group (n = 52) | Allopurinol group (n = 55) | Chuanhutongfeng mixture group (n = 58) | |||
---|---|---|---|---|---|---|
4 weeks | 8 weeks | 4 weeks | 8 weeks | 4 weeks | 8 weeks | |
∆ESR (mm/h) | -8.00 ± 3.80 | -9.00 ± 3.60 | -16.20 ± 9.30 | -17.30 ± 9.20 | -16.40 ± 4.30 | -19.00 ± 4.60 |
∆CRP (mg/L) | -1.20 ± 0. 50 | -2.50 ± 0.50 | -11.80 ± 7.80 | -12.00 ±7.70 | -15.40 ± 8.90 | -15.50 ± 8.90 |
∆Pain score | -1.44 ± 0.51 | -1.55 ± 0.50 | -2.32 ± 0.60 | -2.41 ± 0.53 | -2.36 ± 0.53 | -2.56 ± 0.46 |
∆Swelling score | -1.11 ± 0.42 | -1.15 ± 0.42 | -1.46 ± 0.51 | -1.47 ± 0.41 | -1.47 ± 0.47 | -1.51 ± 0.47 |
∆Restricted movement score | -1.00 ± 0.49 | -1.03 ± 0.49 | -1.47 ± 0.48 | -1.48 ± 0.48 | -1.49 ± 0.50 | -1.53 ± 0.46 |
∆SUA ( | -14.00 ± 12.00 | -26.00 ± 23.00 | -64.00 ± 21.00 | -70.00 ± 21.00 | -90.00 ± 47.00 | -170.00 ± 49.00 |
∆eGFR (ml/min/1.73 m2) | 0.40 ± 2.10 | 0.40 ± 2.90 | -3.00 ± 2.70 | -3.90 ± 3.60 | 3.20 ± 1.70 | 5.70 ± 2.60 |
∆FPG (mmol/L) | -0.05± 0.22 | -0.10 ± 0.22 | -0.10 ± 0.25 | -0.10 ± 0.25 | -0.22 ± 0.32 | -0.20 ± 0.32 |
∆TG (mmol/L) | -0.56 ± 0.37 | -0.58 ± 0.37 | -0.63 ± 0.36 | -0.68 ± 0.35 | -0.61 ± 0.31 | -0.65 ± 0.31 |
∆TC (mmol/L) | -0.22 ± 0.23 | -0.18 ± 0.23 | -0.31 ± 0.22 | -0.36 ± 0.22 | -0.43 ± 0.19 | -0.49± 0.19 |
∆CCL2 (pg/mL) | -21.00 ± 24.00 | -32.00 ± 24.00 | -88.00 ± 76.00 | -163.00 ± 136.00 | -97.00 ± 89.00 | -174.00 ± 129.00 |
∆CXCL8 (pg/mL) | -14.00 ± 18.00 | -21.00 ± 18.00 | -22.00 ± 28.00 | -43.00 ± 38.00 | -93.00 ± 93.00 | -212.00 ± 133.00 |
∆miR-486-5p | 0.12 ± 0.12 | 0.15 ± 0.16 | 0.58 ± 0.53 | 3.28 ± 0.55 | 0.78 ± 0.67 | 5.78 ± 1.72 |
∆miR-339-5p | 0.08 ± 0.16 | 0.11 ± 0.19 | 0.09 ± 0.14 | 1.69 ± 0.47 | 0.60 ± 0.49 | 4.25 ± 1.61 |
∆miR-361-5p | 0.13 ± 0.10 | 0.28± 0.15 | 0.25 ± 0.15 | 4.43 ± 1.06 | 0.63 ± 0.20 | 6.53 ± 2.79 |
The linear correlation analysis of levels of plasma miR-486-5p and miR-361-5p with pain scores and CRP reflecting the patients’ condition are shown in Figure
Analysis of the correlation between levels of plasma miR-486-5p and miR-361-5p, pain scores and CRP in CGA patients. CRP: C-reactive protein; CGA: chronic gouty arthritis.
There was one case (1.72%) of an acute CGA attack in the Chuanhutongfeng mixture group, three cases (5.45%) of recurrence in the allopurinol group, and six cases (11.54%) of recurrence in the control group after 8 weeks of treatment in a PP-analysis. An ITT-analysis revealed five acute CGA attack cases (7.68%) in the Chuanhutongfeng mixture group, eight cases (12.31%) of recurrences in the allopurinol group and eleven cases (16.92%) in the control group. However, both analyses did not show significant differences between the groups (Table
Comparison of acute CGA attack rates in the three groups during the study.
Control group | Allopurinol group | Chuanhutongfeng mixture group | | |
---|---|---|---|---|
PP-analysis (cases/total, %) | 6/52 (11.5%) | 3/55 (5.45%) | 1/58 (1.72%) | 0.096 |
ITT-analysis (cases/total, %) | 11/65 (16.92%) | 8/65 (12.31%) | 5/65 (7.69%) | 0.277 |
CGA: chronic gouty arthritis; PP, per protocol; ITT, intention-to-treat
All symptoms of adverse effects including diarrhea occurred in the control group during the course of the treatment. In the allopurinol group abnormal liver functions with elevated ALT, AST, and bilirubin levels were noted and an inability to tolerate the bitter taste of Chinese medicine and the associated mild diarrhea occurred in one patient of the Chuanhutongfeng group.
We screened gout-specific miRNAs using microarray chips and selected miR-486-5p, miR-339-5p, and miR-361-5p that were differentially expressed in patients with CGA, after which we performed a large sample of RT-qPCR verification. The results showed that the expression levels of miR-486-5p, miR-339-5p, and miR-361-5p in plasma of patients with CGA were significantly lower than in healthy controls. Interestingly, the downregulation of the same three miRNAs in rotator cuff tendinopathy patients indicated concomitant glenohumeral arthritis [
According to previous research, miR-339-5p inhibited the expressions of the proinflammatory factors IL-6, IL-1
The main active component of Chuanhutongfeng mixture is resveratrol, and the existing literature has reported that resveratrol can significantly reduce the inflammatory and apoptotic damage of LPS-induced cells by upregulating miR-146b, thereby inactivating the NF-
We will continue to examine whether resveratrol, the main component of Chuanhutongfeng mixture, can treat CGA by modulating the expression levels of miR-339-5p, miR-486-5p, and miR-361-5p, thus inhibiting the expression of inflammatory factors. In contrast to differential miR-339-5p, miR-486-5p, and miR-361-5p expression pattern, the roles of CCL2 and CXCL8 in gout patients are well known [
However, due to the small number of cases in our study, we need further validation with well-designed, large cohort clinical trials. The downregulation of miR-486-5p, miR-339-5p, and miR-361-5p in plasma may play a role in the pathogenesis of CGA, and upregulation of the expression of miR-486-5p, miR-339-5p, and miR-361-5p may be the mechanism of action(s) of the Chuanhutongfeng mixture and allopurinol in the treatment of CGA.
There are a number of limitations to our research findings. First, the sample size was small and the follow-up time was brief. Second, the mechanism of upregulation of miR-339-5p, miR-486-5p, and miR-361-5p by the Chuanhutongfeng mixture is not fully understood and a direct association of miR-339-5p, miR-486-5p, and miR-361-5p with CCL2 and CXCL8 could not be established. It will be necessary to design a new large cohort clinical study and carry out more in-depth basic research to clarify our findings.
The expression levels of miR-339-5p, miR-486-5p, and miR-361-5p may be associated with CGA. Chuanhutongfeng mixture can significantly upregulate the expression of miR-339-5p, miR-486-5p, and miR-361-5p and reduce the levels of CCL2 and CXCL8. Its clinical efficacy is similar to allopurinol, but with significantly lower side effects. The expression levels of miR-486-5p and miR-361-5p in plasma were correlated with joint pain scores and CRP in patients with CGA and may be used as biological indicators to determine the condition of CGA patients and the likely clinical outcomes.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare no conflicts of interest.
Yao Wang, Peng Liu, and Yangang Wang were responsible for the conception and design of the study. All authors were responsible for the acquisition and analysis of data; furthermore, Yao Wang and Peng Liu were in charge of statistical analysis. Yao Wang drafted the manuscript; Yangang Wang revised and commented on the draft; and all authors read and approved the final version of the manuscript.
This study was supported by the National Natural Science Foundation of China [Grant no. 81571625].