Honokiol-Mesoporous Silica Nanoparticles Inhibit Vascular Restenosis via the Suppression of TGF-β Signaling PathwayHonokiol-Mesoporous Silica Nanoparticles Inhibit Vascular Restenosis via the Suppression of TGF-β Signaling Pathway

doi:10.21203/rs.2.20863/v1

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Honokiol-Mesoporous Silica Nanoparticles Inhibit Vascular Restenosis via the Suppression of TGF-β Signaling PathwayHonokiol-Mesoporous Silica Nanoparticles Inhibit Vascular Restenosis via the Suppression of TGF-β Signaling Pathway

https://doi.org/10.21203/rs.2.20863/v1

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Background and aims: MSNPs improves the solubility of drugs through physical, chemical and other interactions, and can be used to overcome the defects of poor water-solubility of drugs. We used MSNPs standard substance to encapsulate honokiol and then assemble into honokiol-mesoporous silica nanoparticles, and we investigated the effect of these nanoparticles on the process of restenosis after common carotid artery injury in rats.

Results: Here, we report a promising delivery system that load honokiol into mesoporous silica nanoparticles (MSNPs), and finally assemble into a nano composite particle. This HNK-MSNPs not merely inhibit proliferation and migration of VSMCs by reducing phosphorylation of Smad3 but also showed a higher suppression of vascular restenosis than the free-honokiol form in a rat model of balloon injury.

Conclusions: This drug delivery system supplies a potent nano-platform for the intracellular delivery of honokiol to VSMCs and shows a promising use for the future clinical trial.

Nanoscience

Mesoporous silica nanoparticles

Honokiol

Vascular smooth muscle cells

TGF-β pathway

Balloon injury

Restenosis after vascular injury

For patients with multivessel coronary artery disease, percutaneous coronary intervention (PCI) is clinically believed to effectively improve myocardial perfusion [1]. Furthermore, the emergence of drug-eluting stents (DESs) greatly promotes the wide use of PCI. Compared with bare-metal stents (BMSs), application of DESs significantly reduced the incidence of in-stent restenosis (ISR) [2]. However, recurrent lumen narrowing still occurs in many patients after PCI due to the proliferation and migration of excessive vascular smooth muscle cells (VSMCs) [3]. Actually, in many vascular hyperplastic diseases such as hypertension, atherosclerosis and restenosis, VSMCs proliferation is believed to be the main pathological mechanism [4]. After expansible metallic stent implantation, the integrity of vascular endothelium was destroyed due to mechanical injury. Lymphocytes, monocytes and platelets all infiltrate at the sites of damage and produce large amounts of cytokines and inflammatory mediators [5, 6]. Stimulated by cytokines and inflammatory mediators, the phenotype of VSMCs changes from contractile to synthetic type. Active synthetic VSMCs constantly proliferate, migrate and accumulate in the intima and perform repair function, inducing vascular remodeling [7–9].

It was found that the repair process of VSMCs could be accelerated by transforming growth factor-beta (TGF-β) through the activation of multiple intracellular signal pathways [10, 11]. TGF-β is a ligand to cell surface receptor that could activate phosphorylation of smad protein in cells. Smad protein is an important regulatory molecule of TGF-β superfamily signal transduction, in which p-smad2/3 as part of receptor-regulated smads is transported to the nucleus with the assistance of common-mediator smad (p-smad4). In the nucleus, the R-smad/Co-smad complex can initiated the proliferating cell nuclear antigen (PCNA) gene transcription process by interacting with other nuclear cofactor proteins. Furthermore, p-smad2/3 can activate the extracellular signal-regulated kinase (ERK) pathway of mitogen-activated protein kinase (MAPK) family [12–14].

Restenosis after PCI is not only caused by vascular remodeling, but also closely related to the deposition of extracellular matrix (ECM) [10]. Collagen is the main component of ECM, which is secreted by synthetic VSMCs. Excessive secretion result in imbalance of collagen synthesis and degradation [15]. Thus, restenosis is still a clinical challenge to be resolved after interventional therapy. The exploit of novel compounds that can inhibit the proliferation of TGF-β-dependent VSMCs may provide opportunities for treatment of restenosis.

Honokiol (HNK) is one of the main active ingredients in magnolia officinalis. It has been used to treat gastrointestinal disorders, anxiety, neurological disorders and other diseases as a folk medicine for centuries [16].Our previous study demonstrated that free-honokiol can significantly inhibit intimal thickening after common carotid artery injury in rabbits [17]. However, the underlying mechanism remains unclear. And the clinical application was hampered by physiochemical characteristics of honokiol like poor stability due to the oxidation, poor water solubility and bioavailability. Therefore, we tried to design a nano-carrier system for honokiol encapsulation and delivery to overcome these unsatisfying physicochemical properties.

The advent of nanomaterials makes it easier to transport drugs into cells. Nanomaterials can effectively increase the uptake of drugs and thus improve its efficacy, and they may reduce toxicity and side effects of drugs through spatiotemporally controlled release. Ideal nanomaterials are often with good biodegradability, biocompatibility, low toxicity and easiness to produce [18–21]. The mesoporous structure of mesoporous silica nanoparticles (MSNPs) endows its highly uniform pore passage, large surface area, narrow pore diameter distribution and wide range, so MSNPs can accommodate and adsorb small molecule drugs [22–24]. MSNPs improves the solubility of drugs through physical, chemical and other interactions, and can be used to overcome the defects of poor water-solubility of drugs [25]. MSNPs prove to be a good candidate for the present study because (i) it has a high surface area in contact with living organisms, (ii) pores of uniform size make drug diffuse and release constantly, (iii) drug molecules are confined in mesoporous structures to avoid recrystallization [26, 27]. MSNPs is believed to be easily absorbed by cells and has high transmission efficiency. With the decomposition of MSNPs, encapsulated drugs could be released and the carrier fragments are easily excreted through the renal system [20]. Therefore, MSNPs provides an ideal platform for the delivery of small molecule drugs.

Here, we designed a delivery system of honokiol-loaded MSNPs that can successfully transport honokiol into cells and improve the pharmacodynamic effects of honokiol (Fig. 1). We use MSNPs standard substance to encapsulate honokiol and then assemble into honokiol-mesoporous silica nanoparticles (hereinafter referred as “HNK-M”). It was found that HNK-M could inhibit the migration and proliferation of VSMCs, which may be related with the inhibition of TGF-β signaling pathway. Periadventitial application of HNK-M by F-127 pluronic gel demonstrated that HNK-M effectively inhibited the process of restenosis after common carotid artery injury in rats.

Materials

Honokiol was purchased from Selleck Chemicals (Houston, USA), Mesoporous silica nanoparticles and F‑127 pluronic gel was purchased from Sigma‑Aldrich. Transmission electron microscope was acquired from FEI (Hillsboro, USA). Fetal bovine serum (FBS) and 0.25% (w/v) trypsin − EDTA were obtained from Corning (New York, USA). Dulbecco’s modified Eagle’s medium (DMEM) and penicillin − streptomycin solution were purchased from Gibco (Grand Island, NY). Culture flasks (25 cm2 surface area), culture plates, a filter of 8 µm pore size and confocal dishes were acquired from Corning (New York, USA). 2F Fogarty embolectomy catheters were obtained from Edwards Lifesciences (Irvine, CA, USA). WST-1was purchased from Beyotime Biotechnology (shanghai, China). Foxp3/Transcription Factor Staining Buffer Set was purchased from ThermoFisher (Massachusetts, US). The primary antibodies used in western blotting analysis were: anti-p-smad3, anti-p-ERK1/2 and anti-PCNA were purchased from Immunoway Biotechnology (New York, USA). α‑SMA were obtained from Abcam (Cambridge, U.K.).

Ethics Statement

Animals received humane care, the experiment was approved by the Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine, all the animal protocols were carried out in accordance with the Animal Care and Use committee of Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine.

Preparation of the honokiol-loaded Mesoporous Silica nanoparticles (HNK-M)

Honokiol-loaded mesoporous silica nanoparticles (HNK-M) were prepared using a conventional method with modified parameters [28]. Briefly, MSNPs were sonicated and then the mixtures of free-honokiol/MSNPs in 1:2 weight ratio was dispersed in appropriate ethyl alcohol. The complex was left in a shaker for 24 h at room temperature in the dark. The HNK-M was obtained and dried with nitrogen. The residua were concentrated to uniformly disperse in moderate deionized water. When the mesoporous silica carrier decomposes, encapsulated honokiol was release simultaneously. In order to protect the stability of nanoparticles, HNK-M in this study was dried and preserved in the powder form, in which case neither honokiol escaped nor MSNPs decomposition occurred [20].

Characterization of the HNK-M

The morphological shapes of the prepared nanoparticles were observed by transmission electron microscope. The nanoparticles were dispersed with distilled water at 0.5 mg/ml concentration and sonicated in order to form a system with homogenous density. HNK-M was placed on a copper grid coated with carbon membrane and the grid was dried at room temperature before observation.

Cell culture

The vascular smooth muscle cells (VSMCs) obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 µg/ml streptomycin in 75-cm2 flasks at 37 °C in a humidified atmosphere of 5% CO2.

Preparation of the agentia-loaded pluronic gel

F127 hydrogels containing free-honokiol, MSNPS and HNK-M were prepared by the traditional method [17]. 0.3 g of pluronic F127 was dissolved in 1 ml salina overnight at 4℃, and then free-honokiol, MSNPs and HNK-M (contains 2 mg honokiol and 4 mg MSNPs, respectively) were added and vortexed. The mixture was stored at 4℃ for future use.

Construction of balloon injury model and in vivo drug delivery

Male Sprague-Dawley rats (450–550 g) underwent common carotid artery balloon injury using a conventional method [29]. As shown in Fig. 5a-b, the rats were anesthetized with pentobarbital sodium and made an incision in the middle of the neck. A 2-F balloon catheter was inserted into the common carotid artery through the external carotid artery, filled with 50µL normal saline, and pulled three times to establish the animal model of vascular balloon injury. Immediately following balloon injury, the agentia-loaded pluronic gel was applied around the outside of the injured artery. The gel coagulated immediately at body temperature. Animals were euthanized and then the carotid arteries were retrieved 3, 7, 14 and 28 days post‑surgery.

Cytotoxicity assay of free honokiol and HNK-MSNPs

The cytotoxicity of free-honokiol and HNK-M on VSMCs was evaluated with the WST-1 cell proliferation and cytotoxicity assay kit. Briefly, cell suspensions were distributed in a 96-well plate at a density of 1*104 cell/well in 200 µL of standard medium for 24 hours. The cell was then exposed to a series of free-honokiol and HNK-M at different concentrations and incubated at 37 °C with 5% CO2 for 24 h. After the appropriate incubation time, the supernatant was removed and then the mixture with 20 µL of WST-1 solution and 100 µL of standard medium was added to each well. Next, the 96-well plate incubated at 37 °C in 5% CO2 atmosphere for 1.5 h, and the absorbance of each sample was measured in 450 nm using an automated microplate reader. The relative cell viability (%) was estimated from data of three individual experiments, and results were expressed as mean ± SD.

Flow cytometric analysis

The VSMCs were harvested after incubation with the drugs, washed with PBS twice, fixed and permeated using the Foxp3/Transcription Factor Staining Buffer Set. And then, the cells were blocked with blocking solution containing 0.5% BSA/permeabilization buffer for 15 min, stained with the PCNA-antibody. Finally, the samples were washed twice with staining buffer and the cell proliferation assays were examined with FACS Calibur flow cytometer.

Migration assay

For the migration assays, we used transwell inserts with a filter of 8 µm pore size. The VSMCs cultured in serum free system for 24 h and then 6 × 104 cells in serum-free medium and seeded into the supper chamber of the transwell insert with free-honokiol and HNK-M, respectively. The cells were allowed to migrate toward the lower chamber, in which culture medium with 10% FBS was placed. After 24 h incubation, cells in the upper part of the transwell were removed with a cotton swab, and the cells migrated on the lower surface of inserts were fixed by 4% paraformaldehyde and stained with 0.1% crystal violet. Filters were photographed and the total number of cells was counted from 6 randomly selected fields.

Western blot analysis

Following treatment for the indicated conditions, VSMCs and common carotid artery were washed with cold PBS and lysed in RIPA buffer. Cells and tissue were centrifuged at 12000 rpm for 5 min, and the supernatant containing the protein was collected. The concentration of protein was determined using a BCA protein assay kit. 20 µg of protein from each sample were electrophoretically separated using a 12% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel and transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked in 5% BSA for 2 h at room temperature, followed by incubation overnight at 4℃ with the primary antibodies. The following primary antibodies and dilution ratios were used: anti-p-smad3; anti-p-ERK; anti-PCNA. The membranes were rinsed and incubated for 2 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody. The immunoreactive bands were visualized with an enhanced chemiluminescence detection kit. The relative protein levels were quantified by image-pro-plus software version 6.0.

qRT-pcr analysis

Total RNA was harvested from cells using TRIzol reagent and reverse transcribed into cDNA using the SuperScriptIII reverse transcriptase kit. Resultant PCR products were quantified using SYBR Green Master. cDNA samples were adjusted to yield equal GAPDH amplifications. Sequences of the PCR primers are listed in Table 1.

Table 1. Primers used for qRT-PCR.

Target gene Primer sequences

Rat-GAPDH-F (bp518): 5'-GCATCCTGCACCACCAACT-3'

Rat-GAPDH-R (bp618C): 5'-GCAGTGATGGCATGGACTGT-3'

PCNA-rat –F (bp501): 5'-ATATGCCGGGACCTTAGCC-3'

PCNA-rat –R (bp675C): 5'-GCTGAACTGGCTCATTCATCTC-3'

Immunofluorescence staining

VSMCs were allowed to grow on the slides in conditioned culture medium. After incubation by free-honokiol, MSNPs and HNK-MSNPs (80 µM,120 µM), the slides were washed 3 times in phosphate-buffered saline (PBS), fixed in staining fix solution for 15 min, and then permeabilized with 0.5% Triton X-100 in PBS containing 1% BSA for 30 min. After washing with PBS twice, the cells were stained overnight at 4℃ with Alexa Fluor 594-conjugated anti-rat PCNA antibody. The cells were then washed 3 times with PBS, stained with DAPI for 5 min, and observed using a confocal microscopy. In vivo, to analyze the expression of PCNA in the intima, paraffin sections of the common carotid artery were prepared and the VSMCs of intima were located with α-SMC.

Histology and morphometry

The tissue samples were fixed in formalin solution for 24 h and then embedded in paraffin. Then, the samples were cut into 5 µm sections, stained with hematoxylin and eosin (H&E) to detect intima area and intima/media ratio and masson to detect collagen fibers. The tissue stained area was performed by the Image‑Pro Plus version 6.0.

Preparation and characterization of honokiol-loaded MSNPs (HNK-M)

Hydrophobic drug honokiol was encapsulated in hydrophilic silica nanoparticles. The HNK-M compound can improve the efficacy of honokiol and cellular uptake compared to the free-honokiol. MSNPs have hydrophilic surfaces and are easier to be absorbed by cells. Besides, MSNPs are acknowledged to be one of the most promising drug-carrier systems because it can be readily removed from the body and is nontoxic. Small molecule drugs (e.g., honokiol in this study) were usually loaded into MSNPs by three generic strategies, i.e., the molecules of honokiol are embedded into dense interior of MSNPs, encapsulated in the mesoporous structure of MSNPs, or physically adsorbed on the surface of MSNPs [20]. We prepared HNK-M with a conventional method in our laboratory and evaluated its morphology and also the toxicity to VSMCs. Figure 2a showed a typical transmission electron microscopy (TEM) image of HNK-M. They were spherical in shape and superimposed on top of each other. The density inside HNK-M is not uniform as a result of the presence of mesoporous interspace. The WST-1 assay was used for quantitative testing of the viability of VSMCs treated with free-honokiol, MSNPs and HNK-M. WST-1 is a compound similar to MTT and can be reduced to orange formazan by dehydrogenase in the mitochondria. It was shown that the amount of formazan dye is directly correlated to the number of living cells [30]. As shown in Figure 2b, MSNPs is almost nontoxic to VSMCs. The optimal concentration of honokiol for effective therapy on VSMCs was believed to be around 100μM. Figure 2b showed that there was no difference in toxicity between free-honokiol and HNK-M at concentrations ranging from 80μM to 120μM. The IC₅₀ values for free-honokiol and HNK-M were 136.8 and 152.7μM, respectively. These results provided that MSNPs effectively delivers the honokiol to VSMCs by virtue of its preferable biocompatibility and reduce the toxicity of the honokiol to cells. Thus, to avoid potential interference with cell viability, the non-cytotoxic concentrations of honokiol (≤136.8μM) were used in the subsequent experiments.

HNK-M inhibits VSMCs growth in vitro

PCNA is an accessory protein of DNA polymerase delta and is required in the synthesis of DNA during the cell cycle [31]. Thus, PCNA can be used as an indicator to evaluate cell proliferation. In order to evaluate the inhibition effect of HNK on proliferation of VSMCs, flow cytometry was used to determine the expression of PCNA in VSMCs treated with free-honokiol and HNK-M respectively. As shown in Figure 3a-b, the downregulation of PCNA expression in VSMCs treated with MSNPs was similar to that in blank control group, indicating that MSNPs has no inhibitory effects on proliferation of VSMCs. Over the same concentration range, the downregulation rates of PCNA in the free-honokiol group were 7.4% and 14.2% respectively at concentration of 80 and 120μM. However, the results of the HNK-M group were 17.1% and 24.5%, which were both larger than that with corresponding concentrations in free-honokiol group. Taken together, these results suggest that the application of MSNPs significantly increased the ability of honokiol to inhibit the proliferation of VSMCs. In addition, the dependence of PCNA inhibition on concentration of honokiol was also observed both in free-honokiol and HNK-M group.

HNK-M inhibits VSMCs migration

As described above, migration of VSMCs to the intima is one of the mechanisms leading to restenosis after vascular injury. To test whether HNK-M can inhibit the migration of VSMCs, we performed a transwell assay on cultured VSMCs treated with HNK, MSNPs and HNK-M. Transwell assay was carried out by Boyden chamber. It consists of two chambers separated by a microporous membrane as a physical barrier so that cells can only overcome by active migration. As shown in Figure 3c-d, on the average, there were 78.5 cells crossing the membrane in control group. While in both free-honokiol and HNK-M group, fewer migrating cells were observed. When compared with that in free-honokiol group, HNK-M significantly reduced the number of migrating cells both at 80 and 120μM concentration. We also found that HNK-M inhibited VSMCs migration in a concentration-dependent manner. These results confirmed that HNK-M can show an efficient inhibition on VSMCs migration.

HNK-M down-regulates proteins involved in TGF-β signaling pathway

The regulation of TGF-β on VSMCs proliferation is related to a variety of downstream pathways, e.g., the activation of the samd family signaling pathway [32]. To evaluate the potential inhibitory effects on TGF-β signaling pathway, western blots were performed on p-smad3 of VSMCs treated with free-honokiol, MSNPs and HNK-M. As shown in Figure 4a-b, overexpression of p-smad3 on VSMCs was observed by irritation with TGF-β (5ng/ml) for 1h. The phosphorylation state of smad3 can be inhibited by free-honokiol and HNK-M, and the inhibitory effects of HNK-M is more obvious than that of free-HNK. Beyond that, we further evaluated the interaction of smad family and ERK pathway in VSMCs and explored whether HNK-M downregulated the expression of PCNA induced by p-ERK. VSMCs were cultured with SIS3 (SIS3 is a cell-permeable and selective inhibitor of smad3) or PD98059 (PD98059 is a potent, selective and cell-permeable MEK1 and MEK2 inhibitor) and pretreated with free-honokiol, MSNPs and HNK-M for 24h, respectively. The expression of p-ERK and PCNA were measured with western blot assay. In Figure 4c-f, we confirmed that the expression of activated ERK1/2 decreased when inhibiting the phosphorylation process of smad3, and HNK-M can significantly down-regulate the expression of p-ERK1/2 and PCNA compared to the free-honokiol group.

HNK-M reduces PCNA expression in vitro

PCNA, a protein synthesized in early G1 and S phases of the cell cycle, maintains at a high level in proliferation cells [33]. In most cases, signaling pathways promote cell proliferation by inducing PCNA expression. In this study, using RT-PCR, we found that HNK-M reduced the expression of PCNA mRNA in VSMCs when compared with other groups (Figure 5a). To further evaluate the expression of PCNA protein, IF technology was utilized to detect the fluorescence intensity of PCNA in each group. As shown in Figure 5b-c, VSMCs in control group expressed highest levels of PCNA, while free-honokiol and HNK-M groups showed a remarkably lower fluorescence intensity of PCNA. At the same concentration, the fluorescence intensity of PCNA in HNK-M group was lower than that in free-honokiol group, which indicates the significance of MSNPs carrier for HNK delivery.

HNK-M inhibits intimal hyperplasia after vascular injury

In order to determine the point-in-time of draw materials after drug administration to animals, degree of intimal was measured after common carotid artery injury at day 3, 7, 14 and 28. As shown in Figure 6c, there was no visible hyperplasia of intima after 3 and 7 days of vascular injury, so it was impractical to evaluate the changes of intima during this period. The new neointima was developed enough at 14 days after injury, and the neointima formation process kept at a stable state compared with 28 days, this is consistent with previous research [34]. Hence, 14 days after carotid injury operation was set as the predetermined timepoint in the following study to draw materials from the rats.

Intimal hyperplasia is the primary cause of restenosis after vascular injury [35]. The potential of HNK-M in prevention and treatment of restenosis after vascular injury can be preliminarily evaluated by investigating the intimal thickness 14 days after balloon injury. We applied a HE staining to determine the intimal situation in this rat balloon injury model. As shown in Figure 7a, in the control group, the neointimal hyperplasia caused by balloon injury induced significant stenosis of the artery, which almost blocked the lumen. Compared with the control group, HNK-M administration can markedly inhibit the intimal hyperplasia, which was then quantified by increased intima/media ratio and intima area. In figure 7b-c, at 14 days, there was a 72.5% decrease in the intima area of HNK-M group compared with that of free-honokiol group (0.2651±0.024 vs 0.0728±0.009, P=0.0019). Meanwhile, the intima/media ratio decreased by 64.5% (0.1520±0.005 vs 0.0539±0.007, P=0.0004). However, no differences were observed in intimal area between control group and MSNPs group. The results indicated that HNK-M administration had a higher inhibition on the intima hyperplasia in the injured vascular compared with free-honokiol.

HNK-M inhibits collagen deposition

Activation, aggregation and adhesion of platelets were induced at the site of injury after endothelial injury. The activated platelets secrete growth factors and cytokines which then induce VSMCs migration, proliferation and ECM synthesis. This is followed by cellular proliferation and migration and the expression of ECM proteins leading to the formation of neointima and restenosis after stent implantation or angioplasty [15, 36]. Collagen is the main component of ECM and we have previously shown that free-honokiol inhibits collagen deposition in injured carotid artery. The position rate of intimal collagen was measured by masson staining after 14 days of damaged vessels with drug administration. Figure 7d-e revealed that collagen expression was upregulated in the neointima of drug treatment group. However, the deposition of collagen in vascular intima was less in the HNK-M group than the free-honokiol group (P＜0.05), which indicated a higher inhibition of HNK-M on collagen deposition.

HNK-M inhibits VSMCs proliferation in vivo

In normal vascular tissue, VSMCs have a contractile phenotype: they proliferate slowly, are functionally contractile and express a range of contractile proteins, including α-SM-actin (α-SMA) [37] . We investigated whether HNK-M could suppress expression of PCNA and VSMCs proliferation in vivo using a rat carotid artery balloon-injured model. We performed a immunofluorescence staining for α‑SMA and PCNA, and used α‑SMA to localize VSMCs in the intima. As presented in Figure 8a-b, after 14 days of injury, the immunofluorescence of the blood vessels showed that most cells in the neointima expressed α‑SMA (green). PCNA expression in VSMCs of HNK-M treatment group was significantly down-regulated compared with the control group (P=0.0067), and yet, there was no difference between the free-honokiol group and the control group (P=0.0898). These data suggest that HNK-M significantly reduced the proliferation ability of VSMCs in the intima compared to the free-honokiol.

HNK-M inhibits the conduction of the TGF-β signaling pathway in vivo

New data from in vivo and in vitro studies have shown that TGF-β plays an important role in the regulation of a key pathological response to restenosis: intimal thickening and arterial remodeling [38]. We have demonstrated in vitro that TGF-β can stimulate the phosphorylation of smad2/3 in VSMCs, and overexpression of p-smad2/3 can also activate ERK1/2. Therefore, in in vivo experiments, we also used western blot to evaluate the expressions of TGF-β, p-smad2/3 and p-ERK1/2 in common carotid artery tissues at predetermined time after ballon-injuryed carotid artery in rats. As shown in Figure 8c-f, at 3days, 7days and 14days after balloon injury, free-honokiol and HNK-M had no effect on the expression of TGF-β, but inhibited the expression of p-smad2/3 and p-ERK1/2, and the inhibitory effect of HNK-M was slightly stronger than that of the free-honokiol. This result demonstrated that p-smad2/3 and p-ERK1/2 expression was increased from the 3rd day after endovascular injury, and could continue until the 14th day. The in vivo and in vitro results are consistent, further demonstrating that the free-honokiol inhibits VSMCs proliferation by inhibiting the TGF-βpathway, and that the effectiveness of the free-honokiol can be enhanced by encapsulated with MSNPs.

In this study, free-honokiol was encapsulated with MSNPs to get HNK-M, which could effectively deliver honokiol into VSMCs and come into biological effects. Meanwhile, it was found that HNK-M improve the pharmacodynamics of honokiol both in vitro and in vivo. The in vitro results showed that MSNPs was nontoxic to VSMCs and HNK-M can significantly inhibit the proliferation and migration of VSMCs. In vivo experiments showed that HNK-M can effectively inhibit the intimal thickening process after vascular balloon injury in rats, and inhibit the proliferation of VSMCs and deposition of ECM in intima. Studies on in vitro and in vivo anti-proliferation mechanisms indicated that honokiol inhibited the proliferation of VSMCs by inhibiting the conduction of TGF-β signal pathway and the phosphorylation of intracellular related proteins. Taken together, HNK-M delivery system described in this study is a promising candidate for clinical prevention and treatment of restenosis after PCI.

Authors’ contributions

WY conceived the initial idea and the conceptualization. WY and PL designed the study and participated in the data extraction and analysis. XW, YW, ZF, YW and JS participated in study design, searched databases, assessed studies and drafted the manuscript. XW wrote and WY and PL revised the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We appreciate assistance from the faculty of the Instrumental Analysis Center (IAC) of Shanghai Jiao Tong University.

Funding

The work was supported by the National Natural Science Foundation of China (No. 81300092). This study was also partly sponsored by the Interdisciplinary Program of Shanghai Jiao Tong University (No. YG2017MS22, and YG2017QN56) and the Translational Medicine Program of Shanghai Jiao Tong University (No. ZH2018QNA56).

Availability of data and materials

All data generated or analyzed during this study is available from the corresponding author on reasonable request.

Ethics approval and consent to participate

The experiment was approved by the Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine, all the animal protocols were carried out in accordance with the Animal Care and Use committee of Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine.

Consent for publication

Not applicable.

Competing interests

The authors declare no conflict of interest.

Author details

¹Department of Geriatrics, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, No. 639, Zhizaoju Road, Shanghai 200011, China

²Department of Cardiology, Shidong Hospital of Yangpu District, 999 Shiguang Road, Shanghai 200438, China

³Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, & School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China

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Honokiol-Mesoporous Silica Nanoparticles Inhibit Vascular Restenosis via the Suppression of TGF-β Signaling PathwayHonokiol-Mesoporous Silica Nanoparticles Inhibit Vascular Restenosis via the Suppression of TGF-β Signaling Pathway

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Abstract

Figures

Background

Materials and methods

Materials

Ethics Statement

Preparation of the honokiol-loaded Mesoporous Silica nanoparticles (HNK-M)

Characterization of the HNK-M

Cell culture

Preparation of the agentia-loaded pluronic gel

Construction of balloon injury model and in vivo drug delivery

Cytotoxicity assay of free honokiol and HNK-MSNPs

Flow cytometric analysis

Migration assay

Western blot analysis

qRT-pcr analysis

Immunofluorescence staining

Histology and morphometry

Results and Discussions

Conclusion

Declarations

References

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