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Introduction

Chronic kidney disease (CKD) is a long-term condition characterized by renal dysfunction that lasts 3 months (1). CKD remains a leading public health issue, and patients with CKD have poorer quality of life compared with those of healthy individuals (2). There are numerous causes of CKD, the most typical of which are hypertension and type 2 diabetes (3). Although several studies on CKD have been performed (4,5), the molecular mechanisms involved in the occurrence and development of this disease are not fully understood yet. Therefore, there is an urgent need to understand the mechanism of CKD and to identify appropriate strategies to manage it.

Renal interstitial fibrosis occurs in virtually every type of CKD, and the severity of tubulointerstitial fibrosis has long been considered a crucial factor of progressive renal injury in glomerulonephritis (6). The primary histopathological features of renal interstitial fibrosis include marked accumulation of fibroblasts and deposition of extracellular matrix (7). Several cellular pathways, including the mesangial and tubular epithelial-myofibroblast transdifferentiation (TEMT), have been identified as important in renal interstitial fibrosis (8). The main feature of TEMT is the fact that renal tubular epithelial cells acquire mesenchymal phenotypes and myofibroblast functions. The transdifferentiation of TEMT induces kidney epithelial cells to decrease the expression of adherens junction proteins, such as E-cadherin, and to promote the expression of numerous fibroblast markers, including α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1) and fibroblast growth factor-2 (FGF-2) (9). The inhibition of TEMT has also been considered as an effective way to attenuate renal interstitial fibrosis (10).

Heparan sulfate (HS) proteoglycans are important constituents of the cell membrane, and they act as co-receptors for cellular signaling. Cell surface HS proteoglycans are important regulators of cellular migratory, mitogenic, secretory and inflammatory activities (11). Syndecan-1 (SDC1) is a member of the syndecan family, which comprises a transmembrane HS proteoglycan. It can exert various biological functions at the surface of renal epithelial cells, and plays important roles in cell-cell or cell-matrix interactions (12). Heparanase (HPSE), as an endo-β-D-glucuronidase, can cleave HS at specific intrachain sites (13). Precious studies reported that increased levels of HPSE activity are associated with several diseases, such as renal fibrosis, cancer (14) and Alzheimer's disease (15). Several stimulating factors, such as ROS and angiotensin II (AngII), can promote the expression of HPSE, which in turn cleaves the HS side-chains of SDC1 and promotes TEMT, eventually leading to renal fibrosis (16).

Salvianolic acid B (Sal B) is a phenolic acid isolated from Salvia miltiorrhiza Bge. It exhibits various pharmacological activities, including antioxidant (17), anti-myocardial ischemia (18), antitumor (19), and anti-inflammatory (20) activities. A previous study has demonstrated that Sal B has kidney-protective effects (21). However, whether Sal B could reduce renal interstitial fibrosis by regulating the activation of the HPSE/SDC1 axis remains unknown. It was hypothesized that the mechanism of Sal B against renal interstitial fibrosis may be associated with the inhibition of the HPSE/SDC1 axis. Therefore, the present study was designed to investigate the renal-protective effects of Sal B and to explore its underlying mechanism.

Materials and methods

Materials

Sal B (≥98% purity) was provided by Nanjing Hongqiao Institute of Pharmaceutical Technology. Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12) was purchased from Wisent, Inc. Fetal bovine serum (FBS) was purchased from Biological Industries. Trypsin was purchased from Beyotime Institute of Biotechnology. Serum creatinine (CR; cat. no. ANG-E12726H), blood urea nitrogen (BUN; cat. no. ANG-E12727H), TGF-β1 (cat. no. ANG-E10095H) and FGF-2 (cat. no. ANG-E12728H) ELISA kits were purchased from AngleGene BioTechnology Co. Ltd, Nanjing, China. AngII was purchased from Tocris Bioscience. Primary antibodies against HPSE (cat. no. ab59787), SDC1 (cat. no. ab128936), TGF-β1 (cat. no. ab92486), FGF-2 (cat. no. ab8880) and α-SMA (cat. no. ab5694) were obtained from Abcam. Antibodies against E-cadherin (cat. no. 3195S) and GAPDH (cat. no. 5174s) were obtained from Cell Signaling Technology, Inc. The water used in this study was purified by a Milli-Q® water purification system (EMD Millipore). All culture plates were obtained from Corning, Inc.

Animals

A total of 35 2-month-old male C57BL/6 (C57) mice (20±2 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed under pathogen-free conditions under a 12-h light/dark cycle at 22–24°C and had ad libitum access to water and a regular pellet diet (22). The present study was carried out in accordance with the principles of the Basel Declaration and the recommendations of the Guidelines of Jiangsu Regulation for the Administration of Laboratory Animals, and the protocols were approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine (approval no. ACU-2320151203).

Experimental protocol and drug administration

The mice were randomly divided into 5 groups (n=7 mice/group, the group size was decided based on the initial pilot study), including the sham, unilateral ureteral obstruction (UUO), UUO + Sal B (6.25 mg/kg), UUO + Sal B (12.5 mg/kg) and UUO + Sal B (25 mg/kg) groups. The mice in the UUO and Sal B treatment groups were subjected to partial UUO operation according to a previous study (23). Briefly, sodium pentobarbital (50 mg/kg) was used to anesthetize the mice, and then the left ureter was isolated and a 0.20-mm steel wire segment was placed next to the left ureter at the ureteropelvic junction. The wire was ligated with the ureter using a single 6-0 silk suture, and then the wire was removed. The mice in sham group underwent the same operation without ligation. After the operation, the mice in the Sal B treatment groups received a daily intraperitoneal injection of Sal B, while the mice in the sham and UUO groups received equal amounts of normal saline. All mice were sacrificed 14 days later. The anesthetic drug sodium pentobarbital (50 mg/kg) was used to anesthetize the mice, then the blood was collected from the mice retro-orbital sinus for a series of biochemical assays. Then, CO2 (20% V/min; 1.2 l/min) was used to euthanize mice at the endpoint, and the kidneys were harvested for morphological and biochemical studies. Skillful operations were applied to reduce animal suffering during the experiment.

Histopathological observation

The kidneys were immersed in 10% formaldehyde solution for 24 h at room temperature immediately after isolation and then embedded in paraffin. Next, the kidney tissues were cut into 4-µm-thick sections (24). The tissue sections were stained with hematoxylin and eosin (H&E) for 5 min or Masson's stain for 10 min at room temperature to observed the histopathological changes under a light microscope at ×200 magnification.

Immunohistochemical analyses

Mice kidney sections were treated for 10 min with 3% hydrogen peroxide/methanol and 30 min with 5% BSA (Solarbio Life Sciences; cat. no. A8010) at room temperature. Sections were incubated overnight at 4°C with primary antibodies against HPSE (1:100) and SDC1 (1:100), and then labeled with streptavidin-peroxidase (1:50; cat. no. A0208; Beyotime Institute of Biotechnology) for 30 min at 37°C, followed by 5 min 3,3′-diaminobenzidine tetrahydrochloride solution staining, 8 min hematoxylin counterstaining and mounting using neutral balsam. Immunoreactive proteins were viewed with a light microscope at ×200 magnification.

Determination of CR, BUN, TGF-β1 and FGF-2 levels in serum

Blood was collected from the mice retro-orbital sinus on day 14 after surgery. Then, the serum CR, BUN, TGF-β1 and FGF-2 levels were assessed with a microplate system (BioTek Instruments, Inc.) according to the manufacturer's instructions (25).

Cell culture and treatment

HK-2 cells were obtained from American Type Culture Collection and routinely cultured in DMEM/F12 supplemented with 10% FBS and incubated at 37°C with 5% CO2.

HK-2 cells were seeded on a 6-well plate a density of 5×104 cells. Subsequently, 1 µM AngII was added to stimulate the cells for 48 h at 37°C. Then, cells were treated with different concentrations of Sal B (0.1, 1 and 10 µM) for 24 h at 37°C. The cells or the cell supernatants (centrifugation at 300 × g at room temperature for 5 min) were then collected for various analyses.

MTT assay

To determine the appropriate AngII treatment concentration, an MTT assay was performed. Briefly, HK-2 cells were seeded on a 96-well plate at a density of 1×104 cells. After culture overnight, different concentrations of AngII (0, 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 µM) were added to each well and stimulated for 48 h at 37°C. Then, MTT (5 mg/ml) prepared with phosphate buffer (PBS) was added to each well (10 µl). After incubation for 4 h at 37°C, the supernatant of each well was discarded and the formazan was dissolved in 150 µl DMSO. Then, the absorbance at 570 nm was read by BioTek Synergy™ 2 microplate reader (BioTek Instruments, Inc.) and the cell viability of each group was calculated. According to the experimental results, when the concentration of AngII was ≥10 µM, the viability of HK-2 cells declined markedly, and AngII stimulation (1 µM) could notably promote the expression of key regulators of TEMT, such as TGF-β1 and FGF-2. So, 1 µM was selected as the treatment concentration of AngII (data not shown).

Detection of TGF-β1 and FGF-2 in cell supernatant

HK-2 cells were seeded on a 6-well plate at a density of 5×104 cells. After overnight culture, AngII (final concentration, 1 µM) was added to each well and stimulated for 48 h at 37°C. Subsequently, cells were treated with different concentrations of Sal B (0.1, 1 and 10 µM) for 24 h at 37°C, and the cell supernatant of each group was collected for analysis (centrifugation at 300 × g at room temperature for 5 min).

Immunofluorescence

HK-2 cells seeded on glass coverslips in 24-well plates at a density of 2×104 cells were washed 3 times with PBS after treatment, and then fixed for 30 min with 4% paraformaldehyde at 37°C. Next, cells were washed twice with PBS, incubated with 1% BSA (Beijing Solarbio Science & Technology Co., Ltd., cat. no. A8010) for 1 h at room temperature, and incubated with a primary rabbit anti-HPSE (1:100) or anti-SDC1 (1:100) antibody in blocking buffer for 12 h at 4°C. Then, cells were washed twice with PBS and incubated with DAPI-goat anti-rabbit antibody (1:500; Beijing Solarbio Science & Technology Co., Ltd., cat. no. A0562) for 2 h at 37°C. The images were acquired using a fluorescence microscope (26).

Western blotting

The kidney tissues (including both the medulla and cortex) and HK-2 cells were homogenized in ice-cold RIPA buffer containing 2 mM PMSF. The samples were centrifuged at 12,000 × g for 15 min at 4ºC, and the concentrations of total protein were quantified using a NanoDrop instrument (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). Total protein samples (30 µg) were then separated by SDS-PAGE on 6–10% gels and electro-transferred onto PVDF membranes. Then, the membranes were blocked with 5% BSA for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against HPSE (1:1,000), SDC1 (1:1,000), FGF-2 (1:1,000), TGF-β1 (1:1,000), E-cadherin (1:1,000), α-SMA (1:1,000) and GAPDH (1:1,000). After washing 3 times with TBS-Tween-20 (1%), membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:1,000; Beijing Solarbio Science & Technology Co., Ltd., cat. no. A0208) in 5% BSA at room temperature for 1 h (27). The membranes were visualized using an ECL advanced kit and target proteins were detected with a gel imaging system (Image Lab software version 4.0.1; Bio-Rad Laboratories, Inc.).

Cell transfection

Plasmids for overexpression of HPSE and empty control plasmids were purchased from Shanghai GenePharma Co., Ltd. HK-2 cells were seeded on a 6-well plate at a density of 5×104 cells and divided into five groups, including the negative control (NC), Homo HPSE + AngII, Homo HPSE, NC + AngII and Homo HPSE + AngII + Sal B groups. Transient transfection was performed using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 3 µg overexpressed RNA (final concentration, 3 µg/ml), and 9 µl Lipofectamine 2000 were added to the serum-free medium and incubated at 25°C for 20 min. Then, the plasmids were mixed into per group and cultured in serum-free DMEM at 37°C. After 6 h in culture, the medium was replaced by DMEM containing 10% FBS and the cells were cultured for 24 h at 37°C.

Statistical analysis

Data are presented as the mean ± SD. The statistical significance of differences between the means of multiple groups was analyzed by one-way ANOVA, followed by Tukey's multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.

Results

Effect of Sal B on renal morphology in mice with renal fibrosis

To explore the protective effects of Sal B on UUO-induced mice kidneys, 3 mice were randomly selected 14 days after modelling. As shown in Fig. 1, the sizes of the kidney in the UUO group were markedly increased after left ureteral ligation, whereas Sal B treatment notably reduced the kidney sizes.

Figure 1. Effect of salvianolic acid B on the renal morphology of each group of mice (n=7 mice/group). UUO, unilateral ureteral obstruction.

Sal B alleviates the pathological changes of mice kidneys

The results of H&E staining demonstrated that the kidneys of the mice in the sham group maintained a normal morphology and clear structure with no interstitial edema, while those of the mice in the UUO group showed notable cavitation in the renal tubular epithelium, evident inflammatory cell infiltration in the renal tubulointerstitium and distinct tubular expansion. These conditions could notably be restored by Sal B treatment (Fig. 2). Besides, the result of Masson's staining showed that there were numerous collagen fiber streaks stained blue with obvious collagen fiber hypertrophy in the UUO group. However, fewer collagen fiber streaks were observed in the Sal B treatment groups. These results indicated that Sal B could alleviate renal injury and fibrosis in fibrotic mice (Fig. 3).

Figure 2. Histopathological observation of the kidneys in each group of mice (n=3 mice/group). In the sham group, the kidney tissues maintained a normal morphology and clear structure. In the UUO group, the kidney tissues showed marked cavitation in the renal tubular epithelium and evident inflammatory cell infiltration. In the Sal B (6.25 mg/kg) and Sal B (12.5 mg/kg) groups, the kidney tissues showed partial cavitation of the tubular epithelium with moderate inflammatory cell infiltration in the renal tubulointerstitium. In the Sal B (25 mg/kg) group, the tubular epithelial cavitation and inflammatory cell infiltration were notably alleviated (magnification, ×200). Sal B, salvianolic acid B; UUO, unilateral ureteral obstruction.

Figure 3. Representative images of Masson's staining of the kidneys of mice in each group (n=3 mice/group). The UUO group showed numerous collagen fiber streaks with obvious collagen fiber hypertrophy, but these conditions were notably alleviated by salvianolic acid B treatment (magnification, ×200). UUO, unilateral ureteral obstruction.

Effects of Sal B on BUN/CR in the serum of renal fibrotic mice

The serum levels of BUN and CR are the classical markers of renal function (28). To evaluate the efficacy of Sal B on renal function after left ureteral ligation, the expression levels of BUN and CR in serum were determined in the present study. The results showed that left ureteral ligation resulted in a significant increase in the levels of BUN and CR (Fig. 4), but the administration of Sal B (12.5 and 25 mg/kg) notably reduced the expression of these cytokines.

Figure 4. Effects of Sal B on BUN/CR in serum (n=3 mice/group). Mice were intraperitoneally injected with Sal B after left ureteral ligation. Data are expressed as the mean ± SD. *P<0.05 and **P<0.01 vs. UUO group; ##P<0.01 vs. sham group. Sal B, salvianolic acid B; UUO, unilateral ureteral obstruction.

Sal B regulates the expression levels of HPSE and SDC1 in mice kidneys

Using immunohistochemical analysis, the effect of Sal B on the expression of HPSE and SDC1 in kidney tissues was examined. As shown in Fig. 5, left ureteral ligation notably downregulated the expression level of HPSE and upregulated SDC1. However, these changes were notably altered by Sal B treatment.

Figure 5. Immunochemical staining of HPSE and SDC1 (n=3). Salvianolic acid B treatment notably downregulated the expression of HPSE but upregulated the expression of SDC1 in mice kidneys (magnification, ×200). UUO, unilateral ureteral obstruction; HPSE, heparinase; SDC1, syndecan-1.

Sal B inhibits TEMT in renal fibrotic mice

To evaluate whether Sal B could affect the expression levels of fibrosis markers, ELISA and western blotting were performed. The primary feature of renal interstitial fibrosis is the accumulation of extracellular matrix components. Myofibroblasts are considered to be primary renal interstitial cells, which can produce large quantities of extracellular matrix during fibrosis (29). Furthermore, the activation of TEMT is closely associated with the production of myofibroblasts (30). The present results showed that Sal B could significantly downregulate the expression level of α-SMA and upregulate the protein level of E-cadherin compared with the UUO group (Fig. 6). TGF-β1 and FGF-2 are key regulators of the TEMT process (31), the present study demonstrated that Sal B attenuated the UUO-induced upregulation of these proteins in UUO mice (Figs. 6 and 7).

Figure 6. Salvianolic acid B reduces tubular epithelial-myofibroblast transdifferentiation in UUO mice. Western blotting was performed to determine the protein expression levels of FGF-2, TGF-β1, E-cadherin and α-SMA in the kidney tissues of each group of mice. The expression was normalized to that of GAPDH, and the data were expressed as the mean ± SD of 3 independent experiments. *P<0.05 and **P<0.01 vs. UUO group; ##P<0.01 vs. sham group. UUO, unilateral ureteral obstruction; FGF-2, fibroblast growth factor-2; TGF-β1, transforming growth factor-β1; α-SMA, α-smooth muscle actin.

Figure 7. Effect of Sal B on TGF-β1/FGF-2 in serum (n=3 mice/group). Mice were intraperitoneally injected with Sal B after left ureteral ligation. Data are expressed as the mean ± SD. *P<0.05 and **P<0.01 vs. UUO group; ##P<0.01 vs. sham group. Sal B, salvianolic acid B; UUO, unilateral ureteral obstruction; FGF-2, fibroblast growth factor-2; TGF-β1, transforming growth factor-β1.

Effects of Sal B on TGF-β1/FGF-2 in the supernatant of HK-2 cells

TGF-β1 and FGF-2 are key regulators of TEMT (32). To evaluate the efficacy of Sal B on TEMT, the expression levels of TGF-β1 and FGF-2 in HK-2 cell supernatant were determined. The results showed that AngII stimulation significantly increased the expression levels of TGF-β1 and FGF-2 (Fig. 8). However, Sal B treatment (1 and 10 µM) significantly reduced the expression levels of these cytokines.

Figure 8. Sal B significantly reduces the expression levels of TGF-β1 and FGF-2 in HK-2 cell supernatant (n=3). Data are expressed as the mean ± SD. *P<0.05 and **P<0.01 vs. model group; ##P<0.01 vs. control group. Sal B, salvianolic acid B; FGF-2, fibroblast growth factor-2; TGF-β1, transforming growth factor-β1; AngII, angiotensin II.

Sal B suppresses renal interstitial fibrosis by inhibiting the HPSE/SDC1 axis

To explore the underlying mechanisms of Sal B-mediated renal protection, the protein expression levels of the HPSE/SDC1 axis were detected. The results showed that the protein expression of HPSE, FGF-2, TGF-β1 and α-SMA were significantly increased, while the expression levels of SDC1 and E-cadherin were significantly reduced after modelling compared with those of the control group. However, treatment with Sal B significantly reversed these changes (Fig. 9). Furthermore, the results of immunofluorescence also showed that Sal B could notably reduce the expression level of HPSE and promote the expression level of SDC1 in HK-2 cells after treatment with AngII (Fig. 10).

Figure 9. Salvianolic acid B inhibits tubular epithelial-myofibroblast transdifferentiation in HK-2 cells. Western blotting was performed to determine the protein expression levels of HPSE, SDC1, FGF-2, TGF-β1, E-cadherin and α-SMA in each group. The expression was normalized to that of GAPDH and the data are expressed as the mean ± SD of 3 independent experiments. *P<0.05 and **P<0.01 vs. model group; ##P<0.01 vs. control group. HPSE, heparinase; SDC1, syndecan-1; FGF-2, fibroblast growth factor-2; TGF-β1, transforming growth factor-β1; α-SMA, α-smooth muscle actin; AngII, angiotensin II.

Figure 10. Salvianolic acid B treatment notably downregulates the expression of HPSE and upregulates the expression of SDC1 in HK-2 cells (n=3). HPSE, heparinase; SDC1, syndecan-1; AngII, angiotensin II.

Overexpression of the HPSE gene suppresses renal interstitial fibrosis by inhibiting the HPSE/SDC1 axis

In order to further assess the effects of the overexpression of the HPSE gene on renal interstitial fibrosis, the protein expression levels of the HPSE/SDC1 axis were detected using western blotting. The findings showed that overexpression of HPSE could significantly upregulate the expression levels of HPSE, FGF-2, TGF-β1 and α-SMA, and downregulate the expression levels of SDC1 and E-cadherin. However, treatment with Sal B could notably restore these changes (Fig. 11).

Figure 11. Sal B treatment significantly inhibits the activation of the HPSE/SDC1 axis induced by overexpression of the HPSE gene. The expression was normalized to that of GAPDH, and the data are expressed as the mean ± SD of 3 independent experiments. *P<0.05 and **P<0.01 vs. Homo HPSE + AngII group; #P<0.05 and ##P<0.01 vs. NC group. NC, negative control; Sal B, salvianolic acid B; HPSE, heparinase; SDC1, syndecan-1; AngII, angiotensin II; FGF-2, fibroblast growth factor-2; TGF-β1, transforming growth factor-β1; α-SMA, α-smooth muscle actin.

Discussion

Various types of CKDs can lead to renal fibrosis and eventually renal failure; thus, there are numerous studies on the factors behind renal fibrosis. AngII is a key mediator that is associated with the development of renal fibrosis (33,34), it has been reported to promote TGF-β1 expression, leading to the production of extracellular matrix and inhibition of the extracellular matrix degradation (35). Moreover, TGF-β1 has been reported to induce tubular epithelial cells to transdifferentiate into myofibroblasts (36). In the present study, AngII was used to promote the transdifferentiation of HK-2 cells into myoblasts and to promote the expression of TGF-β1. The results showed that AngII could promote TEMT via activation of the HPSE/SDC1 axis. However, Sal B treatment notably decreased the expression levels of extracellular matrix and TEMT markers.

TEMT plays an important role in renal interstitial fibrosis. Under the stimulation of numerous factors, renal tubular epithelial cells could release cytokines (such as TGF-β1 and FGF-2) into the tubulointerstitial microenvironment, and eventually lead to TEMT (37,38), which is characterized by decreased expression of E-cadherin and increased expression of α-SMA (39). Sal B, as one of the main water-soluble components of Salvia miltiorrhiza Bge, has been shown by previous studies to be able to inhibit TEMT by antioxidation (40) and to decrease α-SMA expression (41). The present study demonstrated that Sal B treatment could significantly reduce the expression levels of TGF-β1 and FGF-2, and regulate the expression of E-cadherin and α-SMA.

HS proteoglycans are important constituents of the cell membrane and are associated with multiple physiological processes (42). Previous reports demonstrated that SDC1 in renal tubular epithelial cells is rich in HS chains. HPSE could specifically cleave the HS side chain of SDC1 and lead to the release of several cytokines, such as TGF-β1 and FGF-2. The present study showed that Sal B could significantly reduce the expression of HPSE, which indicates that Sal B could attenuate renal interstitial fibrosis by regulating the HPSE/SDC1 axis. However, considering the characteristics of natural products with multiple targets, further in vitro and in vivo research is required to investigate the underlying mechanisms of Sal B in renal interstitial fibrosis.

In summary, the present study demonstrated that activation of the HPSE/SDC1 axis, which promotes TEMT responses and exacerbates renal injury, may play an important role in renal interstitial fibrosis. Sal B, as a natural product with little toxicity, may provide new prospects for the treatment of renal interstitial fibrosis.

Acknowledgements

Not applicable.

Funding

This study was supported by Jiangsu Province University Brand Professional Construction Project (grant no. PPZY2015A070).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

YH and MW contributed equally to this work. LX designed the study. YH, MW, YP and QL performed the experiments and analyzed the data. YH, MW and LX contributed to the writing of the manuscript. All authors contributed to the revision of this manuscript and approved the final manuscript.

Ethics approval and consent to participate

The protocol was approved by the Animal Ethics Committee of Nanjing University of Chinese Medicine (approval no. ACU-2320151203).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References