Background: Indoxyl sulfate (IS) is a protein-bound uremic toxin with vascular toxicity. The primary cause of death in uremic patients on maintenance hemodialysis is vascular disease, and it had been reported that vascular smooth muscle cells (VSMCs) trans-differentiation (VT) plays a vital role in the context of vascular diseases, but the underlying mechanisms remain obscure. Thrombospondin-1 (TSP-1) participates in vascular calcification by keeping the balance of extracellular matrix, but its role in IS-induced VT is unclear. Methods: In this study, clinical specimens, animal models, and in vitro VSMCs were used to investigate the role of TSP-1 in IS induced VT and the potential therapeutic methods. Results: We found that TSP-1 was significantly decreased in arterial samples from uremic patients, animal models, and in VSMCs after IS treatment. Downregulation of TSP-1 sufficiently induced the trans-differentiation genotypes of VSMCs. Conclusion: Emodin, the main monomer extracted from rhubarb, could alleviate IS-induced VT in vitro by upregulating TSP-1. Taken together, IS induces VT by downregulating TSP-1. Emodin might be a candidate drug to alleviate VT under IS treatment.

Vascular calcification (VC) accompanies a pathological process of atherosclerotic plaque formation, which occurs due to vascular cell trans-differentiation of vascular smooth muscle cells (VSMCs) into cells resembling bone mineralization-competent cells. The medial layer of the vessel wall, where VSMCs trans-differentiation (VT) mainly occur, is composed of extracellular matrix (ECM) and VSMCs. VSMCs are essential for the optimal function of blood vessels and synthesizing ECM molecules [1]. ECM components, such as collagens and con-collagens proteins, influence the function, activity, and the VT [2]. Collagens can represent either pro- or anti-calcifying molecules. These effects are associated with the Ca/P-induced osteochondrocytic VT that exhibits altered expression of different markers (i.e., RUNX2, SOX9, OSX, α-smooth muscle actin [α-SMA], and SM-22α) [3], further highlighting that changes in the composition/characteristics of the ECM environment can modulate VSMC phenotype [4].

Thrombospondin-1 (TSP-1), encoded by the THBS1 gene, is a macromolecular glycoprotein that is widely distributed in the body and is an important factor in regulating ECM homeostasis and mediating cell-ECM dialogue [5‒7]. TSP-1 participates in ECM remodeling by regulating the balance of matrix metalloproteinase (MMPs)/tissue inhibitors of metalloproteinase (TIMPs) [8]. In addition, TSP-1 has been reported to act as an inhibitory regulator of osteoblastic bone mineralization and matrix production to maintain bone homeostasis in mouse osteoblastic MC3T3-E1 cells [9]. TSP-1 is expressed in the thickened intima of atherosclerotic lesions [10, 11] and balloon-injured arteries, where it may regulate smooth muscle cell proliferation and migration [11, 12]. During bone formation, TSP-1 is localized to the unmineralized osteoid and TSP-1 levels decrease as the matrix becomes mineralized [13, 14]. Despite the obvious parallels between the mineralization processes in bone and vascular lesions [10, 15], the association of TSP-1 with vascular trans-differentiation has not been well investigated.

Indoxyl sulfate (IS) affects various signal pathways in VSMCs, such as oxidative stress, inflammation, cellular phenotype, and cell survival, and enhances VC [16, 17]. But the association with IS and TSP-1 in VT is little reported.

In order to explore the above problems, we determined the expression changes of TSP-1 using arterial samples from uremic patients and uremic animal models. Next, the VSMCs were treated with IS to establish an in vitro VT model, and the role of TSP-1 in IS-induced VT was explored through gene interference and therapeutic drug treatment.

Human Artery Samples

Human radial arteries were collected from end-stage renal disease (ESRD) patients undergoing arteriovenous fistula formation for hemodialysis (n = 2). The radial arteries harvested as grafts from age- and sex-matched coronary heart disease patients undergoing coronary artery bypass graft were included as control (n = 2). Human artery collection was performed at Zhongshan Hospital, Shanghai Medical College, Fudan University, Shanghai, China. Ethical approval was obtained from the Clinical Research Ethical Committee of Zhongshan Hospital, Shanghai Medical College, Fudan University, and the methods were carried out in accordance with the approved guidelines. Each patient provided written informed consent to the use of their tissues for research purposes.

Animals and Treatments

All animal experiments were performed following the regulations of the Shanghai Animal Experiment Management Committee. Eight clean male SD rats (6–8 weeks old, weighing 200 ± 20 g) were from Shanghai Shrek Experimental Animal Co., Ltd. The CKD rat model was established by two-step 5/6 nephrectomy (Nx). Blood was collected from the tail vein of rats at the 4th, 8th, and 12th weeks to detect the levels of serum creatinine and serum phosphorus.

Histological and Immunohistochemical Staining

Vascular sections were processed into 5 μm thick sections and stained with H&E or Alizarin Red staining, according to standard histological procedures. The ECM was orange stained and the cells were lavender stained under a light microscope, which was positive for calcium deposition. Vascular sections (5 μm) were deparaffinized and rehydrated, then washed in 100%, 95%, 85%, 75% ethanol, ddH2O, and placed in sodium citrate antigen retrieval solution for antigen retrieval. Sections were incubated with anti-TSP-1 antibody (1:100, Invitrogen, MA5-13377) at 4°C overnight and then incubated with secondary antibody for 1 h at room temperature. DAB kit (1:300, GK500710, Gene Tech) was used to detect the positive signals. Nuclei were stained with DAPI. Sections were washed with PBS and then stained, and then the neutral resin was added to cover the sections. The sections were stored at room temperature, observed under a microscope, and photographed for record.

Cell Culture and Treatment

Primary rabbit aortic VSMCs were isolated from the abdominal aorta of an 8-week-old male New Zealand rabbit. VSMCs were maintained in low glucose DMEM medium (D5030, Sigma-Aldrich) containing 10% fetal bovine serum supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin at standard cell culture conditions (37°C, 5% CO2, 100% humidity). The medium was replaced every 2 days and the cell passaged every 4 days. All experiments were performed with VSMCs in passages 3–8. VSMCs were treated with 1,000 μM of IS for 72 h to induce vascular trans-differentiation [18]. Emodin was added to the medium with different final concentrations (0.5 µM, 1 µM or 5 µM). For TSP-1 knockdown, VSMCs were transfected with TSP-1 siRNA (sense 5′-GCG​UGU​UUG​ACA​UCU​UUG​AAC-3′ and antisense 5′-UCA​AAG​AUG​UCA​AAC​ACG​CUG-3′; negative control siRNA sense, 5′-AAA-3′ and antisense 5′-AAA-3′), and gene expression was measured at 12 h after transfection.

Quantitative Real-Time PCR

Total RNA from kidney tissues of 5/6Nx rats and VSMCs were extracted by Trizol reagent (T9424, Sigma) according to the manufacturer’s protocol. The concentration and quality of RNA were detected by the 260/280 nm ratio using a NanoDrop Spectrophotometer (Thermo). PrimeScript RT Master Mix (RR036B, Takara) was used to generate cDNA from RNA, and quantitative real-time PCR was performed using TB Green Master Mix (RR820B, Takara) and QuantStudloTM 5 Real-Time PCR Instrument. All samples were analyzed in duplicate, and all quantitative data from qRT-PCR were normalized to ACTB (Actin). Sequences of primers are given in Table 1.

Table 1.

Sequences of primers for real-time PCR

Forward (5′-3′)Reverse (5′-3′)
R_Actb CTA​AGG​CCA​ACC​GTG​AAA​AG TAC​ATG​GCT​GGG​GTG​TTG​A 
R_THBS1 TCG​GGG​CAG​GAA​GAC​TAT​GA AGC​CTG​CAG​TTG​TCT​CTG​TC 
Rab_Actb AAG​TGT​GAC​GTT​GAC​ATC​CG TCT​GCA​TCC​TGT​CAG​CAA​TG 
Rab_Acta2 TAC​AAT​GAG​CTT​CGC​GTT​GC TTT​TCT​CCC​GGT​TGG​CTT​TG 
Rab_THBS1 ATG​CTG​GCA​ATG​GCA​TCA​TC TGC​AGT​GGT​AAG​TTG​CGT​TG 
Rab_OPN AGA​CCC​TCC​CGA​GTA​AGT​CC GTG​ACT​TTG​GGT​TTC​CAC​GC 
Rab_NFYA ACA​AAT​TCA​GCA​GCA​GCA​GC TGT​CCA​CTG​CTG​GTT​GTG​TT 
Rab_RUNX2 AAG​GCA​CAG​ACA​GAA​GCT​TG AGG​AAT​GCG​CCC​TAA​ATC​AC 
Rab_ALP ACC​ACC​ACG​AGA​GTG​AAC​CA CGT​TGT​CTG​AGT​ACC​AGT​CCC 
Rab_MMP1 AAA​ATT​ACA​CGC​CAG​ATT​TGC​C GGT​GTG​ACA​TTA​CTC​CAG​AGT​TG 
Rab_MMP9 TGT​CAT​CCA​GTT​TGG​GGT​CG ACT​CTT​TGC​CCA​GGA​AGA​CG 
Rab_TIMP1 AGA​GTG​TCT​GCG​GAT​ACT​TCC CCA​ACA​GTG​TAG​GTC​TTG​GTG 
Rab_TIMP2 AAG​CGG​TCA​GTG​AGA​AGG​AAG GGG​GCC​GTG​TAG​ATA​AAC​TCT​AT 
Rab_TIMP3 AAC​TCC​GAC​ATC​GTG​ATC​CG TTG​ATG​GTC​GTC​TTG​TCC​GG 
Forward (5′-3′)Reverse (5′-3′)
R_Actb CTA​AGG​CCA​ACC​GTG​AAA​AG TAC​ATG​GCT​GGG​GTG​TTG​A 
R_THBS1 TCG​GGG​CAG​GAA​GAC​TAT​GA AGC​CTG​CAG​TTG​TCT​CTG​TC 
Rab_Actb AAG​TGT​GAC​GTT​GAC​ATC​CG TCT​GCA​TCC​TGT​CAG​CAA​TG 
Rab_Acta2 TAC​AAT​GAG​CTT​CGC​GTT​GC TTT​TCT​CCC​GGT​TGG​CTT​TG 
Rab_THBS1 ATG​CTG​GCA​ATG​GCA​TCA​TC TGC​AGT​GGT​AAG​TTG​CGT​TG 
Rab_OPN AGA​CCC​TCC​CGA​GTA​AGT​CC GTG​ACT​TTG​GGT​TTC​CAC​GC 
Rab_NFYA ACA​AAT​TCA​GCA​GCA​GCA​GC TGT​CCA​CTG​CTG​GTT​GTG​TT 
Rab_RUNX2 AAG​GCA​CAG​ACA​GAA​GCT​TG AGG​AAT​GCG​CCC​TAA​ATC​AC 
Rab_ALP ACC​ACC​ACG​AGA​GTG​AAC​CA CGT​TGT​CTG​AGT​ACC​AGT​CCC 
Rab_MMP1 AAA​ATT​ACA​CGC​CAG​ATT​TGC​C GGT​GTG​ACA​TTA​CTC​CAG​AGT​TG 
Rab_MMP9 TGT​CAT​CCA​GTT​TGG​GGT​CG ACT​CTT​TGC​CCA​GGA​AGA​CG 
Rab_TIMP1 AGA​GTG​TCT​GCG​GAT​ACT​TCC CCA​ACA​GTG​TAG​GTC​TTG​GTG 
Rab_TIMP2 AAG​CGG​TCA​GTG​AGA​AGG​AAG GGG​GCC​GTG​TAG​ATA​AAC​TCT​AT 
Rab_TIMP3 AAC​TCC​GAC​ATC​GTG​ATC​CG TTG​ATG​GTC​GTC​TTG​TCC​GG 

Western Blotting Analysis

Total proteins were extracted from VSMCs by Trizol regent (T9424, Sigma) according to the manufacturer’s protocol, subjected to SDS-PAGE, and electrophoretically transferred to 0.45-µm polyvinylidene fluoride membranes. Membranes were placed in blocking solution for 1 h at room temperature and incubated overnight at 4°C with anti-TSP-1 antibody (1:1,000, Cell Signaling Technology, 37879S), anti-α-SMA antibody (1:1,000, Abcam, ab5694), anti-runt-related transcription factor 2 (RUNX2) antibody (1:1,000, Abcam, ab76956), anti-TIMP3 antibody (1:1,000, Abcam, ab85926), anti-MMP9 (1:1,000, Cell Signaling Technology, 3852), anti-Beta-Tubulin (1:1,000, Sino Biological, 100109-MM05T). After washing with TBS-T for three times, the membrane was incubated with the corresponding secondary antibody for 1 h at room temperature. After washing with TBS-T for three times, protein expression was visualized using the ECL ultrasensitive luminescence solution (Tanon, 180-5001B). The intensity of the protein band was quantified by ImageJ.

Statistical Analysis

All data from experiments were presented as mean ± standard errors. Statistical significance was calculated using the unpaired Student’s t test. And the difference was considered statistically significant at p value <0.05. GraphPad Prism (version 9.4.1) was used for statistical analyses.

TSP-1 Was Dramatically Decreased in trans-Differentiated Vascular Smooth Muscle Cells from ESRD Patients and 5/6Nx Rats

Compared to the healthy controls, the radial arteries from ESRD patients showed thickening arterial wall with obvious trans-differentiation assessed by Alizarin red staining (Fig. 1a). Similarly, trans-differentiation of radial arteries was also observed in 5/6Nx rats compared to the sham group (Fig. 1b). At the same time, a great reduction of TSP-1 protein expression in the medial layer of radial arteries was observed in ESRD patients and 5/6Nx rats by immunohistochemistry analysis (Fig. 1c, d). Moreover, the mRNA level of TSP-1 was also significantly lower in the arteries tissue of the CKD rats (online suppl. Fig. 1; for all online suppl. material, see https://doi.org/10.1159/000532028).

Fig. 1.

TSP-1 was dramatically decreased in trans-differentiated vascular smooth muscle cells from ESRD patients and 5/6Nx rats. Visualization of trans-differentiation in radial artery sections. Representative H&E and Alizarin red-stained radial artery sections of an ESRD patient and 5/6Nx rat (a, b). H&E and Alizarin red staining results showed that compared with the internal mammary artery of the control group, the elastic layer of the radial artery in the uremic patients (×200 magnification, bar = 50 µm) and the 5/6Nx rats (×400 magnification, bar = 25 µm) was lightly stained, the structure was disordered, and the quantification of the trans-differentiation also showed the calcium deposition in the arterial media was significantly increased. c, d Immunohistochemical staining and quantification showed that compared with the artery of the control group, the expression of TSP-1 in the radial artery of the uremic patients (×200 magnification, bar = 50 µm) and the 5/6Nx rats (×400 magnification, bar = 25 µm) was significantly reduced.

Fig. 1.

TSP-1 was dramatically decreased in trans-differentiated vascular smooth muscle cells from ESRD patients and 5/6Nx rats. Visualization of trans-differentiation in radial artery sections. Representative H&E and Alizarin red-stained radial artery sections of an ESRD patient and 5/6Nx rat (a, b). H&E and Alizarin red staining results showed that compared with the internal mammary artery of the control group, the elastic layer of the radial artery in the uremic patients (×200 magnification, bar = 50 µm) and the 5/6Nx rats (×400 magnification, bar = 25 µm) was lightly stained, the structure was disordered, and the quantification of the trans-differentiation also showed the calcium deposition in the arterial media was significantly increased. c, d Immunohistochemical staining and quantification showed that compared with the artery of the control group, the expression of TSP-1 in the radial artery of the uremic patients (×200 magnification, bar = 50 µm) and the 5/6Nx rats (×400 magnification, bar = 25 µm) was significantly reduced.

Close modal

TSP-1 and α-SMA Were Significantly Decreased in IS-Induced VT in vitro

Compared to the control group, α-SMA, the marker gene of VSMCs, was significantly decreased both at mRNA (Fig. 2a) and protein levels (Fig. 2b) in VSMCs that treated with IS (1,000 μM) for 72 h. The mRNA levels of alkaline phosphatase (ALP) and RUNX2 were greatly upregulated (Fig. 2a), while RUNX2 protein levels showed no significant changes (Fig. 2b). The mRNA level of TIMP3 was downregulated, while the protein expression of TIMP3 and MMP9 did not change. In addition, the gene and protein expression levels of TSP-1 were significantly reduced (Fig. 2a, b), it indicated that VSMCs showed obvious genotype trans-differentiation and downregulation of TSP-1 after IS treatment.

Fig. 2.

TSP-1 and α-SMA were significantly decreased in IS-induced VT in vitro. The mRNA and protein expression levels of smooth muscle related molecules in control and IS group, analyzed by quantitative RT-PCR and Western blotting, respectively. a Real-time PCR results of TSP-1, Acta2, ALP, RUNX2, TIMP3, and MMP9 in IS group compared with control (n = 3). b Western blotting analysis and quantitative results of the characteristic of VSMCs and trans-differentiation (n = 3). Individual bands were quantified gray values and normalized to Tubulin.

Fig. 2.

TSP-1 and α-SMA were significantly decreased in IS-induced VT in vitro. The mRNA and protein expression levels of smooth muscle related molecules in control and IS group, analyzed by quantitative RT-PCR and Western blotting, respectively. a Real-time PCR results of TSP-1, Acta2, ALP, RUNX2, TIMP3, and MMP9 in IS group compared with control (n = 3). b Western blotting analysis and quantitative results of the characteristic of VSMCs and trans-differentiation (n = 3). Individual bands were quantified gray values and normalized to Tubulin.

Close modal

Downregulation of TSP-1 Induces VT

To dig out the role of TSP-1 in VT, siRNA was used to knock down TSP-1 in VSMCs (Fig. 3a). We found that, compared to the negative control, the mRNA and protein expressions of α-SMA were significantly downregulated in TSP-1 siRNA group (Fig. 3a, b). However, knocking down of TSP-1 could not evoke either ALP or RUNX2 expression (Fig. 3a, b). It suggested that knocking down of TSP-1 was sufficient to induce VT in vitro. In addition, we found that the combination of TSP-1 knockdown and IS produced stronger trans-differentiation phenotypes than either treatment alone (online suppl. Fig. 2).

Fig. 3.

Downregulation of TSP-1 induces VT. a The mRNA expression levels of TSP-1 and Acta2 were significantly decreased compared with control group (n = 3). b Western blotting results reveal that TSP-1 and α-SMA were extremely downregulated, while there was no statistical difference in the calcification marker RUNX2 (n = 3). Blots shown are representative examples of data collected from at least three different blots extracted from three independent protein extractions.

Fig. 3.

Downregulation of TSP-1 induces VT. a The mRNA expression levels of TSP-1 and Acta2 were significantly decreased compared with control group (n = 3). b Western blotting results reveal that TSP-1 and α-SMA were extremely downregulated, while there was no statistical difference in the calcification marker RUNX2 (n = 3). Blots shown are representative examples of data collected from at least three different blots extracted from three independent protein extractions.

Close modal

Emodin Ameliorates IS-Induced VT

It has been reported that Emodin (1,3,8-trihydroxy-6-methyl anthraquinone), the main monomer extracted from rhubarb, has a wide activity in anti-cardiovascular diseases in uremia patients [19], and it demonstrated that Emodin attenuates and stabilizes atherosclerotic plaques [20]. We determined the effect of Emodin in IS-induced VT. VSMCs were treated with IS and with or without Emodin. We found that Emodin (0.5 μM and 1 μM) could significantly reverse the downregulation of TSP-1 and α-SMA after IS treatment (Fig. 4a, b), while could not inhibit RUNX2 and ALP (Fig. 4a). However, when VSMCs treated with high concentration of Emodin (5 μM), the expression of RUNX2 was significantly decreased than that of IS-only treated group, and TSP-1 was still significantly increased while out α-SMA upregulation (Fig. 4a), so we selected 1 μM of Emodin for the following experiments. Compared to control group, Emodin could also significantly upregulate TSP-1 and α-SMA in protein level but could not inhibit RUNX2 (Fig. 4b). Moreover, when VSMCs treated with Emodin alone, there was no change in the expression of TSP-1, α-SMA, ALP, and RUNX2 (Fig. 4c). These results indicated that Emodin can inhibit VT, but not calcification, induced by IS.

Fig. 4.

Emodin ameliorates IS-induced VT. The mRNA expression levels (a) and Western blotting results (b) of VSMCs with Emodin in different concentrations under 1,000 μM IS (n = 3). mRNA levels of TSP-1, Acta2, RUNX2, and ALP were robustly increased in the 0.5 μM and 1 μM Emodin treated groups, and protein levels of TSP-1 and α-SMA were upregulated under 1 μM Emodin. c Quantitative real-time PCR analysis showed that TSP-1, Acta2, ALP, and RUNX2 mRNA did not change significantly in VSMCs treated with only 1 μM Emodin (n = 3).

Fig. 4.

Emodin ameliorates IS-induced VT. The mRNA expression levels (a) and Western blotting results (b) of VSMCs with Emodin in different concentrations under 1,000 μM IS (n = 3). mRNA levels of TSP-1, Acta2, RUNX2, and ALP were robustly increased in the 0.5 μM and 1 μM Emodin treated groups, and protein levels of TSP-1 and α-SMA were upregulated under 1 μM Emodin. c Quantitative real-time PCR analysis showed that TSP-1, Acta2, ALP, and RUNX2 mRNA did not change significantly in VSMCs treated with only 1 μM Emodin (n = 3).

Close modal

The Protective Effect of Emodin on IS-Induced VT Was Mediated by TSP-1

Next, we investigated whether the protective effect of Emodin on IS-induced VT was mediated by TSP-1. First, VSMCs transfected with TSP-1 siRNA for 12 h, and then treated with or without 1 μM Emodin for 24 h. We found that Emodin could reverse the downregulation of TSP-1 which was induced by siRNA transfection, at the same time, α-SMA was also significantly upregulated (Fig. 5a, b). However, the expression of RUNX2 was slightly decreased in the Emodin treated groups (Fig. 5a). It indicated that Emodin could reverse the downregulation of TSP-1 either treated by TSP-1 siRNA (Fig. 5a) or IS treatment (Fig. 4a) but has no effect on TSP-1 expression under normal condition (Fig. 4c). Whereas, when VSMCs transfected with TSP-1 siRNA and then treated with both IS and Emodin, the results showed that TSP-1 and Acta2 were significantly decreased compared to control group, it indicated that Emodin cannot reverse the VT under IS treatment (Fig. 5c) when TSP-1 was knocked down.

Fig. 5.

The protective effect of Emodin on IS-induced VT was mediated by TSP-1. a Under the condition of TSP-1 siRNA transfection, Emodin can upregulated TSP-1 and Acta2, and the expression of RUNX2 was also slightly decreased in the Emodin treated groups (n = 3). b Western blotting results showed that the expression of α-SMA and TSP-1 was significantly increased in the 1 μM Emodin group (n = 3). c On the basis of siRNA, VSMC was treated with IS and Emodin together, and the results showed that the expression of TSP-1 and Acta2 was significantly reduced compared to the control group.

Fig. 5.

The protective effect of Emodin on IS-induced VT was mediated by TSP-1. a Under the condition of TSP-1 siRNA transfection, Emodin can upregulated TSP-1 and Acta2, and the expression of RUNX2 was also slightly decreased in the Emodin treated groups (n = 3). b Western blotting results showed that the expression of α-SMA and TSP-1 was significantly increased in the 1 μM Emodin group (n = 3). c On the basis of siRNA, VSMC was treated with IS and Emodin together, and the results showed that the expression of TSP-1 and Acta2 was significantly reduced compared to the control group.

Close modal

It is well known that uremic toxins can induce VC and VT [21], and mounting evidence has confirmed that IS mediates VC by inhibiting Notch pathway, inducing osteogenic differentiation and apoptosis in VSMCs [6, 22]. Other study found that uremic toxin promoted the VC by activating the JNK/Pit-1 pathway [23]. In our study, TSP-1 plays a key role in IS-induced VT and mediates the protective effect of Emodin on IS-induced VT.

α-SMA protein is expressed in VSMCs and myoepithelial cells, and it is also considered a marker of VSMCs [24]. The expression of α-SMA decreased with the transition of VSMCs from the contractile state to the proliferative state [25]. The gene and protein levels of α-SMA were downregulated in VSMCs differentiated into atherosclerosis phenotype induced by oxidative stress [26]. Therefore, among various trans-differentiation markers, α-SMA is considered an excellent marker for the phenotype transition and trans-differentiation of VSMCs [27, 28]. In this study, the expression of α-SMA was closely related with TSP-1, which means that TSP-1 plays a key role in VSMCs phenotype maintenance.

As a widely used anthraquinone natural product, Emodin has been reported to have therapeutic effects on various diseases [29]. It has been proven to have anti-inflammatory, antiapoptotic, anti-fibrosis, antitumor, and other effects and plays a protective role in ischemia-reperfusion injury in multiple organs [30]. Some studies have shown that IS or Emodin can regulate gene expression at both the transcriptional and posttranscriptional levels. First, at the transcriptional level, IS can promote CpG methylation of Klotho gene and repress its expression [18]. Emodin can regulate H3K27 methylation and acetylation modifications and promote cardiac Sirt3 mRNA and protein expression [31]. Second, at the posttranscriptional level, IS downregulates the m6A level of mRNA by increasing the demethylase FTO [32], and IS can also promote endoplasmic reticulum stress and regulate protein degradation [33]. Emodin regulates the protein degradation process by inhibiting the ubiquitination process [34]. In this study, IS can significantly reduce the mRNA and protein expression of TSP-1, and when co-treatment with siRNA, the mRNA of TSP-1 further decreased. This suggests that IS could reduce TSP-1 expression both at the gene and transcription levels. In all of the Emodin treatment groups of this study, Emodin only had an inhibitory effect on TSP-1 reduction under reduced TSP-1 conditions (siRNA or IS treatment), while under normal conditions, Emodin did not affect the mRNA level of TSP-1. Therefore, Emodin may exert an inhibitory effect on TSP-1 mRNA degradation at the posttranscriptional level. And the schematic representation of Emodin inhibiting IS-induced VT by upregulating TSP-1 is presented in Figure 6. In addition, this study found that either in the cases of TSP-1 knockdown, IS and Emodin co-treatment, or siRNA and Emodin co-treatment, there were no obvious changes in the gene and protein expression of TIMP3 and MMP9 (online suppl. Fig. 3), while the changes of α-SMA were significant at that time, indicating that TSP-1 may not regulate ECM through TIMPs/MMPs.

Fig. 6.

Schematic representation of Emodin inhibiting IS-induced VT by upregulating TSP-1.

Fig. 6.

Schematic representation of Emodin inhibiting IS-induced VT by upregulating TSP-1.

Close modal

There were several limitations in our study. The mechanism of Emodin in inhibiting VT has not been verified in animal models. In addition, the cell model of our study was performed only in rabbit-derived VSMCs, and human VSMCs should be further included to make our conclusions more representative. In conclusion, we explored TSP-1 as a key mediator in IS-induced VT, and Emodin inhibits VT through TSP-1 activation.

The investigation was approved by the Ethics Committee of Zhongshan Hospital, Fudan University (Approval No.: B2021-346R).

The authors have no conflicts of interest to declare.

This work was supported by grants from National Nature Science Foundation of China (No. 81803880, No. 81800596), Shanghai “science and technology innovation plan” Yangtze River Delta scientific and technological Innovation Community project (No. 21002411500), Shanghai “science and technology innovation plan” technical standard project (No. 19DZ2205600), Shanghai Federation of Nephrology Project supported by Shanghai ShenKang Hospital Development Center (No. SHDC2202230), and Youth Science Foundation of Zhongshan Hospital, Fudan University (No. 2021ZSQN52).

Shuan Zhao, Xiaoqiang Ding, and Shi Jin were responsible for the conception and design of this study. Jing Chen provided VSMCs, and Weidong Zhang was responsible for experimental data analysis and manuscript writing. Shi Jin and Shuan Zhao were responsible for overall supervision and critical review of the manuscript. All authors participated in the experimental effort of this study. Shi Jin is the first corresponding author.

Data used in this study are available by contacting the corresponding author.

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