Inhibitory Effect of β-Sitosterol on the Ang II-Induced Proliferation of A7r5 Aortic Smooth Muscle Cells

Objective To explore the effects of β-sitosterol on VSMC proliferation. Materials and Methods A7r5 cells were pretreated with 2 µM angiotensin II (Ang II) for 24 hr to establish an excessive VSMC proliferation model, followed by treatment with β-sitosterol for 24 hr. Cells were divided into five groups: control, Ang II, and Ang II + β-sitosterol (2, 4, 8 µM). CCK-8 assay, flow cytometry, and Ad-mCherry-GFP-LC3B assay analyzed cell proliferation, cell cycle, cell apoptosis, and autophagic flux. Additionally, the expression of proteins was detected by the western blotting. Results β-Sitosterol effectively inhibited Ang II-induced A7r5 cell proliferation (IC50 : 6.841 µM at 24 hr). It achieved this by arresting cell cycle progression, promoting apoptosis, inhibiting autophagy, and suppressing the contractile–synthetic phenotypic switch. Mechanistically, β-sitosterol downregulated PCNA, Cyclin D1, and Bcl-2, while upregulating pro-caspase 3, cleaved-caspase 3, and Bax to induce cell cycle arrest and apoptosis. Additionally, it suppressed the contractile–synthetic phenotypic transformation by downregulating OPN and upregulating α-SMA. The Ad-mCherry-GFP-LC3B Assay and western blotting revealed β-sitosterol's autophagy inhibitory effects by downregulating LC3, ULK1, and Beclin-1 while upregulating P62 expression. Discussion and Conclusion. This study found for the first time that β-sitosterol could inhibit the proliferation of A7r5 cells induced by Ang II. β-Sitosterol treatment may be recommended as a therapeutic strategy to prevent the cardiovascular diseases.


Introduction
Vascular smooth muscle cells (VSMCs), a critical component of tunica media, play an irreplaceable role in regulating vascular tone, allowing vascular remodeling, and providing structural support [1][2][3].Excessive proliferation of VSMCs contributes to cardiovascular diseases, including atherosclerosis, aneurysms, and intimal restenosis [4,5].Thus, the development of novel agents to inhibit the excessive proliferation of VSMCs is urgently required to prevent cardiovascular diseases.
Various cytokines and growth factors, including insulin, angiotensin II (Ang II), and platelet-derived growth factors, stimulate excessive proliferation of VSMCs [6][7][8].Ang II, an important peptide in the renin-angiotensin system, stimulates the proliferation of VSMCs by regulating cell cycle progression and apoptosis [9].A recent study found that Ang IIinduced proliferation of VSMCs was related to the regulation of autophagy [10].
Phytosterols are natural plant components structurally similar to cholesterol and are enriched in traditional Chinese herbal medicines, vegetable oils, and plant seeds [11,12].The previous studies have shown that phytosterols and their derivatives protect the cardiovascular system by reducing the absorption of dietary cholesterol [13][14][15].Animal studies have shown that feeding mice with 2% mixed phytosterols can reduce tumor size, inhibit cancer cell growth, and significantly reduce the incidence of prostate, breast, and colon cancer [16,17].In vitro studies have also demonstrated that phytosterols, particularly β-sitosterol, could inhibit the proliferation and induce apoptosis in osteoblasts and various cancer cells [18][19][20].Furthermore, stigmasterol can interrupt the progression of the cell cycle in rat aortic smooth muscle cells (RASMCs) [9].However, the effect of β-sitosterol on VSMC proliferation remains unknown.Thus, we studied the effect of β-sitosterol on the proliferation of A7r5 model cell lines induced by Ang II and explored the related mechanisms.

2.2.
Cell Culture and Treatment.The A7r5 cells were procured from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).A7r5 cells were seeded in DMEM containing 1% penicillin/streptomycin and 10% FBS and cultured at 37°C in an incubator with 5% CO 2 .Third-to-sixth generation A7r5 cells were used in this experiment.After reaching 70%-80% confluence, A7r5 cells were washed with PBS and cultured in a medium with and without Ang II and β-sitosterol at different concentrations for 6-24 hr.Concentrations of β-sitosterol and Ang II were determined as described previously [20,21].

Assessment of Cytotoxicity and Cell Proliferation via
CCK-8 Assay.The CCK-8 was used to evaluate the cytotoxicity of β-sitosterol in A7r5 cells.The assay was repeated five times.A7r5 cells were seeded in 96-well plates at a density of 5 × 10 3 cells/well at room temperature.After cell culture overnight, each well of A7r5 cells was cultured in a medium with and without Ang II (2 µM) and β-sitosterol (0.5, 1, 2, 4, 8 µM) for 24 hr.Subsequently, 10 µl WST-8 solution was added and cultured in an incubator for 1-2 hr, and A7r5 cells in 96-well plates were detected using a microplate absorbance reader at 450 nm.

Assessment of Cell Cycle and Cell Apoptosis via Flow
Cytometry.The cell cycle of A7r5 cells was determined using flow cytometry.In brief, cells were seeded into 6-well plates and then fixed in 70% cold ethanol following treatment with Ang II or β-sitosterol.Cells were then washed with PBS, resuspended in PBS containing ribonuclease, and then stained with propidium iodide in the dark.The fluorescence intensity of the cells (1 × 10 4 cells in each sample) was quantified by the flow cytometer (FACSCalibur™ cytometer, BD Biosciences, USA).
A7r5 cells were assessed for apoptosis using the Annexin V-FITC Kit in accordance with the manufacturer's instructions.A7r5 cells were trypsinized, washed twice with cold PBS, resuspended in 500 μl binding buffer containing 5 μl annexin V-FITC and 5 μl PI, and cultured at room temperature in the dark for 15 min.Finally, cell apoptosis was detected using flow cytometer (FACSCalibur™ cytometer, BD Biosciences, USA).

Assessment of Autophagic
Flux via Ad-mCherry-GFP-LC3B Assay.Induction of autophagic flux was assessed using the Ad-mCherry-GFP-LC3B assay.A7r5 cells were seeded in 6-well culture plates at a density of 2 × 10 4 cells per well until the cells reached 40-50% confluency.Cells were transfected with Ad-mCherry-GFP-LC3B at an MOI of 20 for 24 hr according to the protocol.After that, the cells were treated with β-sitosterol, Ang II or 3-MA for another 24 hr and their morphology was observed under a fluorescence microscope (Leica, Germany).
2.6.Western Blot.Treated A7r5 cells were lysed in lysis buffer (Tris-HCl, NaCl, EDTA, 10% glycerol, 0.1% NP-40, PMSF, aprotinin, leupeptin, NaF, Na3VO4, and DTT) and incubated for 30 min on ice.The supernatant was collected by centrifugation at 12,000 rpm for 10 min at 4°C.A BCA Protein Assay Kit was used to determine the total protein concentration in the supernatant.Proteins from A7r5 cells were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto PVDF membranes.The PVDF membranes were blocked with 5% nonfat milk for 2 hr and incubated with primary antibodies overnight at 4°C, followed by goat anti-rabbit secondary antibodies for 2 hr at room temperature.The PVDF membranes were visualized using a chemiluminescence detection kit, and quantitative analyzes were performed by densitometry using ImageJ 6.0 software (National Institute of Health, Bethesda, Maryland, USA).Analytical Cellular Pathology expression compared to the Ang II-alone treatment group (Figures 2(c) and 2(d)).These results suggest that β-sitosterol inhibits the Ang II-induced phenotype switching from a contractile to synthetic state in A7r5 cells.

Cell Cycle Arrest in
Ang II-Induced A7r5 Cells by β-Sitosterol.Cell cycle progression was examined by flow cytometry to confirm whether β-sitosterol could inhibit the proliferation of Ang II-induced A7r5 cells.As shown in Figure 2(b), treatment of A7r5 cells with Ang II decreased the proportion of cells in the G0/G1 phase from 65.74% AE 1.02% to 54.89% AE 1.43% (P <0:01) and increased the proportion of cells in the S phase from 19.93% AE 1.46% to 33.49% AE 1.34% (P <0:001) compared to the control group.The expression levels of PCNA and Cyclin D1 were also examined.PCNA and Cyclin D1 expression levels were found significantly upregulated in Ang II-alone treatment groups (Figures 2(c) and 2(d)).However, treatment with 2, 4, and 8 µM of β-sitosterol increased the proportion of A7r5 cells in the G0/G1 phase to 58.70% AE 1.88% (P <0:05), 67.20% AE 2.39% (P <0:01), and 67.74% AE 1.97% (P <0:001), and decreased the proportion of A7r5 cells in the S phase to 28.85% AE 2.25% (P <0:05), 17.70% AE 1.84% (P <0:01), and 17.13% AE 3.11% (P <0:001) compared with the Ang II-alone treatment group.Additionally, the expression levels of PCNA and Cyclin D1 were significantly downregulated in the β-sitosterol treatment groups compared with those in the Ang II-alone treatment group.The proportion of A7r5 cells in the G2/M phase in the Ang II alone treatment

4
Analytical Cellular Pathology group and the β-sitosterol treatment group did not differ from that in the control group.These results indicated that β-sitosterol increased the proportion of Ang II-induced A7r5 cells in the G0/G1 phase and decreased the proportion of these cells in the S phase.The expression of autophagy-related proteins (LC3, ULK1, P62, and Beclin-1) was analyzed using western blotting to confirm whether Ang II-induced autophagy at the cellular level was ameliorated by β-sitosterol.Western blot assays revealed increased LC3-II/I ratio, Beclin-1, and ULK1 protein expression levels, and decreased P62 expression levels in the Ang II-alone treatment group (Figures 4(c)-4(e)).However, this effect was reversed by treatment with β-sitosterol (2, 4, and 8 µM) and 3-MA.The inhibitory effect of β-sitosterol was related to its concentration, and the most prominent inhibitory effect was observed at 8 µM.The above results showed that β-sitosterol could inhibit autophagy in Ang II-induced A7r5 cells.

Reversal of β-Sitosterol Effects on Ang II-Induced A7r5
Cells Autophagy and Proliferation by Autophagy Activator Rapamycin.Rapamycin is used to investigate the role of autophagy in the inhibitory effect of β-sitosterol on Ang IIinduced proliferation of A7r5 cells.The β-sitosterol-induced reduction in Ang II-induced proliferation at 8 µM was restored by treatment with 100 nM rapamycin (Figure 5(f)).Next, we wanted to confirm whether protein expression in Ang II-induced A7r5 cells could be influenced by rapamycin.Western blotting results showed that the decreased protein expression of PCNA in Ang II-induced A7r5 cells by 8 µM β-sitosterol was increased by rapamycin administration (Figures 5(c) and 5(d)).
We also observed alterations in autophagic flux and changes in the protein expression of LC3 and P62.Treatment of Ang II-induced A7r5 cells with 8 µM β-sitosterol resulted in a decrease in the LC3-II/I ratio, a reduction in the number of yellow and red dots, and an increase in P62 expression.Meanwhile, the LC3-II/I ratio increased and the expression of P62 decreased, while the number of yellow and red dots increased after cotreatment with β-sitosterol and rapamycin (Figures 5(a)-5(e)).These results showed that β-sitosterol inhibited the proliferation of Ang II-induced A7r5 cells by the downregulation autophagy.

Discussion
Clonal A7r5 embryonic rat thoracic aortic smooth muscle cells retain many characteristics of VSMCs and are thus considered a suitable model of VSMCs [22].In this study, we have experimentally demonstrated that pretreatment with β-sitosterol exerts a remarkable protective effect on A7r5 cells by inhibiting Ang II-induced cell proliferation.Furthermore, for the first time, we identified that β-sitosterol regulates Ang II-mediated apoptosis, cell cycle, autophagy, and the contractile-synthetic phenotypic switch, which may participate in the antiproliferation process of β-sitosterol against Ang II in A7r5 cells.
Numerous studies have clearly demonstrated that Ang II, an important regulator of vascular remodeling, plays a crucial role in the occurrence and development of the cardiovascular diseases [23].In this study, we found that Ang II induced the contractile-synthetic phenotypic switch and proliferation of VSMCs in vitro.Contractile and synthetic are two phenotypes of VSMCs.The transition from the contractile to synthetic form results in the proliferation of VSMCs in hypertension [24].Uncontrolled proliferation, migration, and matrix synthesis of VSMCs are critical cytopathological links in the process of vascular remodeling in hypertension and atherosclerosis [25,26].In hypertension, vascular remodeling involves dynamic structural and morphological changes that narrow the blood vessels, thicken their walls, increase vascular resistance and pressure, and worsen hypertension and its complications [27].Vascular remodeling in atherosclerosis exacerbates the vulnerability of atherosclerotic plaques, thereby increasing the risk of cardiovascular events [28].In this study, we also found that different concentrations of β-sitosterol alleviated Ang IIinduced A7r5 cell proliferation and inhibited the switch from a contractile to synthetic phenotype in A7r5 cells induced by Ang II.These findings suggest that β-sitosterol holds promise as a therapeutic option for vascular disorders, including hypertension and atherosclerosis.
The eukaryotic cell cycle consists of four phases: G0/G1, S, G2, and M. VSMCs generally exist in a nonproliferative state (G0/G1) under normal conditions.However, in case of       6 Analytical Cellular Pathology blood vessel injuries, Ang II, PDGF-bb, and other stimulatory cytokines and growth factors can induce VSMCs to transform from the nonproliferative state (G0/G1) to the proliferative state (S phase) [29].The present study found a similar cell cycle progression in Ang II-induced A7r5 cells.
After treatment of A7r5 cells with Ang II, the proportions of cells in the G0/G1 phase decreased, and the proportions of cells in the S phase increased.The previous research has demonstrated that β-sitosterol possesses the ability to impede the proliferation of human leukemia cells by inducing cell cycle arrest at G2/M phases [30].Our study found that β-sitosterol significantly decreased the proportion of A7r5 cells in the S phase and increased the proportion of A7r5 cells in the G0/G1 phase.The cell cycle is a complex process that involves multiple regulatory proteins [31].Cyclin D1, an important cyclin involved in the cell cycle regulation, plays a key role in transforming the G1 phase into the S phase [32].The upregulation of Cyclin D1 protein expression can significantly accelerate the transition from the G1 phase to the S phase, causing excessive cell proliferation and tumor formation [32][33][34].PCNA is a nuclear protein involved in the S phase and is an accessory protein that mediates DNA polymerase replication of the DNA of cells [35,36].Blockade of the protein expression of Cyclin D1 and PCNA could significantly reduce the proliferation of aberrant VSMCs in certain pathological events [37].In the present study, Ang II upregulated the protein expression of Cyclin D1 and PCNA in A7r5 cells, which aligns with the previous findings in VSMCs [20].However, treatment with β-sitosterol reduced the protein expression of Cyclin D1 and PCNA in Ang II-induced A7r5 cells.These results suggest that β-sitosterol inhibits Ang II-induced A7r5 cell proliferation by blocking cell cycle progression.Notably, there is strong evidence that Ang II can inhibit the apoptosis of mouse aortic smooth muscle cells and lead to vascular remodeling [38][39][40].In the present study, A7r5 cells treated with Ang II exhibited a significantly lower proportion of apoptotic cells compared to the control group.The previous studies have shown that β-sitosterol induces apoptosis and inhibits the proliferation of ovarian cancer cells.Similarly, our study also showed that β-sitosterol can induce apoptosis in A7r5 cells stimulated by Ang II.Apoptosis is a form of programed cell death that effectively removes unwanted host cells to maintain homeostasis.Bcl-2 and Bax genes are important regulators of apoptosis in the apoptosis signal transduction pathway [41].These genes have opposite roles in apoptosis regulation.Caspase 3, the most critical factor in apoptosis downstream of the caspase cascade, catalyzes the cleavage of specific proteins in cells [42].Bcl-2 and Bax can activate caspase 3 to initiate cell apoptosis by releasing cytochrome c and other factors through the mitochondrial pathway.Cleaved-caspase 3 is an activated form of caspase 3 [43].In the current study, 2 µM Ang II inhibited the apoptosis of A7r5 cells primarily by downregulating the protein expression of pro-caspase 3, cleaved-caspase 3, and Bax, and upregulating the protein expression of Bcl-2.Similar results were reported in the other studies [44].β-Sitosterol has been reported to promote apoptosis in human gastric cancer SGC 7901 cells and human leukemia U937 cells by upregulating the expression of cleaved-caspase 3 and Bax proteins, while downregulating the expression of Bcl-2 protein [12].In the present study, treatment with Autophagy has gained increasing attention as an evolutionarily conserved mechanism that is associated with a variety of cellular signaling pathways and plays a crucial role in the survival and function of VSMCs [45,46].It has been reported that autophagy contributes to Ang II-induced VSMC hypertrophy through an AT1R/RhoA/Rho kinase-dependent mechanism [7].In this study, we found that Ang II induces autophagy in A7r5 cells.Additionally, β-sitosterol has the potential to enhance nonsmall cell lung cancer treatment by inhibiting autophagy [47].Our study showed that β-sitosterol inhibits Ang II-induced autophagy in A7r5 cells.ULK1 is a gathering point for multiple signaling pathways that regulate autophagy initiation [48].A previous study has reported that ULK kinase promotes the recruitment of Beclin-1 to the endoplasmic reticulum, thereby driving the formation of autophagosomes [49].Beclin-1, a class III phosphatidylinositol 3-kinase component, plays a significant role in transport processes mediated by the inner membrane, including autophagosome transport [50].Additionally, overexpression of Beclin-1 increased the levels of ATG4, ATG5, and LC3-II proteins in tongue squamous cell carcinoma cell lines [51].LC3-II, the lipidated form of LC3, is an autophagosomal marker in mammals and is often used to study autophagy in the cardiovascular diseases.P62 acts as a bridge between LC3 and ubiquitinated proteins during autophagosome formation.It gets selectively encapsulated into autophagosomes and subsequently degraded by proteases in autolysosomes, leading to an inverse correlation between P62 expression and autophagic activity [52].In our study, Ang II induced autophagy in A7r5 cells primarily by increasing the ratio of LC3II/I and by enhancing the protein expression of Beclin-1 and ULK1, while downregulating the expression of P62.Similar results have been reported in the previous studies on cardiomyocytes [53].β-Sitosterol supplementation significantly inhibited autophagy in Ang II-induced A7r5 cells, with a concomitant decrease in LC3, Beclin-1, and ULK1 expression, and a concurrent upregulation of P62 expression in A7r5 cells.We found, for the first time, that β-sitosterol could reverse Ang II-induced autophagy in A7r5 cells.
Several studies have demonstrated that inhibition of autophagy contributes to a reversible reduction in cell proliferation in various cell lines, including VSMCs and macrophages [54].Cortistatin protects VSMCs from Ang II-induced proliferation by inhibiting autophagy by regulating SSTR3 and SSTR5 receptors [10].Overexpression of miR-145 ameliorates the proliferation and migration of TGF β1-induced VSMCs by inhibiting autophagy activation [55].In the present study, β-sitosterol significantly inhibited Ang II-induced autophagy and proliferation of A7r5 cells, which could be reversed by the autophagy activator rapamycin.Thus, we speculate that the inhibitory effect of β-sitosterol on the excessive proliferation of Ang IIinduced A7r5 cells is also related to the regulation of autophagy.However, considering gene expression profiles from Ang IItreated A7r5 cells which are used in the different laboratories, the variations derived from the different cell types should be of concerned.There are also differences between different

Conclusion
This study found for the first time that β-sitosterol inhibited Ang II-induced proliferation of A7r5 cells by inducing cell cycle arrest, promoting apoptosis, inhibiting autophagy, and suppressing the contractile-synthetic phenotypic switch.These findings indicate that the administration of β-sitosterol has broad developmental potential as a therapeutic strategy to prevent cardiovascular diseases.

3. 2 .FIGURE 1 :
FIGURE 1: Effect of Ang II and β-sitosterol on A7r5 cell viability.(a) The cytotoxic effect of Ang II on A7r5 cells was detected using the CCK-8 assay.(b) The cytotoxic effect of β-sitosterol on A7r5 cells was detected using the CCK-8 assay.(c) 3D chemical structure of β-sitosterol.Data indicate the mean AE SD (n = 5, n: number of the sample).Significant differences in different groups compared with the control group (without treatment with Ang II or β-sitosterol) are expressed as * P <0:05, * * P <0:01, and * * * P <0:001.

ðaÞðcÞFIGURE 2 :
FIGURE 2: Effects of β-sitosterol on the proliferation, cell cycle progression, and phenotypic switch of Ang II-induced A7r5 cells.(a) Effects of β-sitosterol on the proliferation of Ang II-induced A7r5 cells were detected using the CCK-8 assay.(b) The cell cycle progress of A7r5 cells was tested by flow cytometry.(c) Protein expression of α-SMA, OPN, PCNA, and Cyclin D1 was examined by western blotting.(d) α-SMA, OPN, PCNA and Cyclin D1 protein development gray value analysis histogram.Data indicate the mean AE SD (n = 5, n: number of the sample).Significant differences in different groups compared with the control group (without treatment with Ang II or β-sitosterol) are expressed as * P <0:05, * * P <0:01, * * * P <0:001.Significant differences in different groups compared with the Ang II-alone treatment group are expressed as # P <0:05, ### P <0:01, ### P <0:001.

3. 5 .
Enhancing Effect of β-Sitosterol on the Apoptosis of Ang II-Induced A7r5 Cells.Flow cytometry was performed after annexin V-PITC/PI double staining to investigate whether βsitosterol is involved in the apoptosis of A7r5 cells.As shown in Figures3(a) and 3(b), treatment of A7r5 cells with Ang II decreased the proportion of apoptotic cells to 2.01% AE 0.34% (P <0:001) compared with the control group.Meanwhile, the Ang II-alone treatment group showed significantly downregulated expression of pro-caspase 3, cleaved-caspase 3, and Bax, and upregulated expression of Bcl-2 (Figures3(c) and 3(d)).However, apoptosis of Ang II-induced A7r5 cells increased after β-sitosterol treatment (P <0:05).This effect of β-sitosterol was also confirmed by the upregulated expression levels of pro-caspase 3, cleaved-caspase 3, and Bax, as well as the downregulated expression of Bcl-2.These results indicated that β-sitosterol could promote the apoptosis of Ang IIinduced A7r5 cells.3.6.Inhibitory Effect of β-Sitosterol on the Autophagy of Ang II-Induced A7r5 Cells.Abnormal autophagy in VSMCs is an important pathogenic process in the cardiovascular diseases.We determined whether β-sitosterol protects Ang II-induced A7r5 cells against excessive proliferation by anti-autophagy and examined changes in autophagic flux.The Ad-mCherry-GFP-LC3B assay of A7r5 cells showed that the number of yellow and red dots significantly increased after treatment with Ang II, and β-sitosterol reduced the number of yellow and red dots in Ang II-induced A7r5 cells in a concentrationdependent manner (Figures4(a) and 4(b)).Meanwhile, the number of yellow and red dots also decreased in the 3-MA treatment group.