A Treatment Combined Prussian Blue Nanoparticles With Low-intensity Pulsed Ultrasound Alleviates Cartilage Damage in Knee Osteoarthritis by Initiating PI3K/Akt/mTOR Pathway

Background: Reactive oxidative stress (ROS) related apoptosis in chondrocytes and extracellular matrix (ECM) degradation play crucial roles in the process of osteoarthritis (OA). Prussian blue nanoparticles (PBNPs) are known to scavenge ROS in cellular. Low-intensity pulsed ultrasound (LIPUS) has been used as a non-invasive modality for the is widely used in clinical rehabilitation management of OA. Methods: In this study, we aim to investigate the effects of PBNPs/LIPUS combined treatment on knee osteoarthritis (KOA) and to determine whether phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway mediates this process. Use LPS to process primary cells of knee joint cartilage to establish a cartilage knee arthritis model. After treated with LIPUS and PBNPs, cell viability was rated by CCK-8 and ROS levels were assessed by DCFH-DA. Cell apoptosis was estimated by ow cytometry and TUNEL staining. Articular pathological changes were observed by naked eyes, H&E, and Safranin O staining, then monitored by cartilage lesion grades and Mankin’s score. Protein levels of cleaved caspase-3, Bcl-2, Bax, p-PI3K, PI3K, p-Akt, Akt, p-mTOR, mTOR, IL-1β, MMP3, MMP13, p-JINK, JINK, p-c-Jun, and c-Jun were subjected to western blot. Results: Cellular ROS, apoptosis rate, and TUNEL staining of chondrocytes were fairly decreased in the PBNPs group and the LIPUS group but drastically down-regulated in the PBNPs/LIPUS combination treatment group when compared with the LPS group. Both PBNPs and LIPUS decreased the grades of cartilage lesions and Mankin scores of knee articular cartilage, while combined administration achieved more reduction. Western blot results showed that the cleaved caspase-3, Bax, IL-1β,


Characterization of PBNPs
The particle size and morphology of the PBNPs obtained above were characterized by transmission electron microscopy (TEM, FEI TECNAI G2 F20, FEI, USA), scanning electron microscopy (SEM, FEI Nova Nano 450, FEI, USA) and X-ray diffractometer (PANalytical X'Pert PRO, PANalytical, Holland). Both the average size and the average zeta potential of PBNPs were measured by using a Nano-size potentiometer (Zetasizer Nano ZS90, Malvern, UK).
Cell culture New Zealand rabbits were purchased from Chongqing Medical University and sacri ced after air injection through the ear vein. The knee articular cartilage was separated under aseptic conditions; the fascia and cartilage membrane of the wrapped cartilage tissue was stripped off and placed in a dish containing PBS solution. The isolated cartilage tissue was cut into blocks of 0.3-0.5 mm, transferred into a 25 cm 2 culture ask and washed with PBS solution containing double antibodies 3 times. 0.25% of trypsin was added at a dosage of 10-15 times the volume of cartilage, and digestion was terminated after being treated at 37°C for 1-2 h. Cartilage tissue was incubated with 0.02% type II collagenase at 37°C overnight. Cells were observed using an inverted microscope. In the absence of single cells, DMEM medium was added to dilute the type II collagenase to terminate digestion. The cell mass was blown into a single cell and then ltered with a 200 mesh sieve to collect the ltrated. Next, the ltrated was centrifuged at 1500 r/min for 5 min. After that, the supernatant was discarded, and cells were cultured with DS medium at 37℃ with 5% CO 2 . All the animals were euthanatized according to the standards of the Ethics Committee of Chongqing Medical University.

Animals
Twenty-ve male New Zealand rabbits (11 months old, 3.0-3.5 kg) were obtained from Chongqing Medical University and housed in individual cages with a cycle of 12 h of light and 12 h of darkness at 20-25℃. To construct the KOA model, the anterior cruciate ligament was transected from the right knee of 20 rabbits under general anesthesia (3% pentobarbital, 1 mL/kg) by using ophthalmic scissors as previously described [34]. The other 5 rabbits served as controls. After postoperative wk 1, rabbits were induced to move for 30 min daily for 5 d per wk for 7 wk to promote OA development.
After 4 wk of OA induction [35], 20 rabbits with KOA were randomly divided into 4 groups including KOA (n=5), KOA+PBNPs (120 μg/mL article injection once a week for 6 weeks, n=5), KOA+LIPUS (n=5) and KOA+PBNPs combined with LIPUS (n=5) groups. Rabbits in all groups were sacri ced by air embolization at 6 wk after each intervention. All experiments were approved by the Animal Management Rule of the Chinese Ministry of Health and the Chongqing Medical University Animal Ethics Committee.

Macroscopic observation
The femoral condyle articular cartilages from knee joints were collected 6 wk after ACLT and observed by naked eyes. The criterion of cartilage injury grading was referred to as Outerbridge's grading standard [36] ( Table 1).

Histopathology
After general observation, the specimens were xed with neutral formalin and processed for histopathologic examination. The samples were decalci ed in ethylenediaminetetraacetic acid for 3 wk, embedded in para n, and sliced into 4 μm sections using a microtome for microscopic examination. As for the rabbits with KOA, LIPUS was administrated to the right knee as follows: acoustic intensities of 60 mW/cm 2 , a duty ratio of 20%, central frequency of 1.5 MHz, repetition frequency of 1 kHz, the irradiation time of 20 min per day, and 5 days per week for 6 wk. The process was standardized with a device that the rabbits were placed in a supine position with the knee angled approximately 120 at the exion position. The ultrasound probe was attached to the skin of the medial femoral condyle, and the target tissue was cartilage of the medial femoral condyle.

Cell death assessment
The effects of PBNPs on cartilage cell death were determined by Cell Counting Kit-8 (CCK-8) colorimetric assay (Sigma, USA). Brie y, podocytes were seeded into 96-well plates at a density of 10 4 cells per well and cultured in complete RPMI-1640 culture medium for 24 h. Then, the cells were treated with PBNPs at a concentration of 0, 30, 60, 120, 150, and 180 μg/mL respectively for 24 h and 120 μg/mL for 1, 2, 3, 4, 5, 6 and 7 days. CCK8 was added to each well and the cells were incubated at 37°C with 5% CO 2 for 2 h.
Absorbance was quanti ed at 450 nm using a multi-well uorescent plate reader (Thermo Scienti c Varioskan Flash, Thermo Fisher Scienti c, USA). The rate of cell death was calculated.

Flow cytometric analysis
The treated cells were collected by centrifugation at 1000 r/min for 3 min, washed twice with ice-cold PBS, gently resuspended in 500 μL 1×Annexin V binding buffer containing 5 μL Annexin V-FITC and 3 μL of PI before being incubated at room temperature in the dark for 10 min. The percentage of apoptotic cells was analyzed by ow cytometry (BD FACSCalibur, Becton-Dickinson, USA).

Tunel staining
Cell apoptosis was determined using the One-Step TdT-mediated dUTP Nick-End Labeling (TUNEL) Apoptosis Assay Kit (Beyotime, China). Pretreated cells in 12-well plates were washed twice with PBS, incubated with 50 μL TUNEL testing solution at 37℃ for 1 h in the dark, and then washed with PBS 3 times. After the membrane was sealed with anti-uorescence quenching solution, the cells were observed at an excitation wavelength of 550 nm and the emission wavelength of 570 nm using a uorescence microscope (Zeiss Fluorescence Microscope, Germany).

ROS detection
The pretreated cells in 12-well plates were incubated with 2', 7-dichloro uorescein diacetate (DCFH-DA) . Protein blot images were captured using an ECL chemiluminescence system (GE Healthcare, Piscataway, NJ, USA) and quanti ed with Quantity One software.

Statistical analysis
Data were presented as mean±SD from 3 independent experiments. Analyses were carried out by using GraphPad Prism Software version 6.00 (San Diego, CA). The data complied with normal distribution and homogeneity of variance. Statistical comparisons between the two groups were analyzed using the twotailed unpaired Student's t-test. Differences among multiple groups were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey's t-test. The value of P less than 0.05 was considered statistically signi cant.

Characterization of PBNPs
The PBNPs were prepared by using simple colloidal chemistry with polyvinylpyrrolidone (PVP), which is a universal auxiliary material in pharmaceuticals as a stabilizer. The morphology of PBNPs was observed by using scanning electron microscopy (SEM) (Fig. 1A) and transmission electron microscopy (TEM) (Fig. 1B). The synthesized PBNPs were cubic particles with an average size of about 80.06 nm (Fig. 1C) and the average zeta potential of PBNPs was about -15.1 mv (Fig. 1D). The single-crystal structure was con rmed by electron diffraction of the whole particle of PBNPs (Fig. 1E).
The cell viability assay was performed with different concentrations of PBNPs, and no signi cant difference was revealed between the groups of 30 μg/mL, 60 μg/mL, and 120 μg/mL and the control group (incubated with normal saline). Conversely, a remarkable decrease in the cell viability was exhibited in the groups of 150 μg/mL and 180 μg/mL (Fig. 1F). Moreover, no signi cant difference was shown from day 1 to day 7 when the cells were treated with 120 μg/mL PBNPs at a different time (Fig. 1G).

Effects of PBNPs/LIPUS treatment on the ROS induced by LPS in chondrocytes
To investigate effects of PBNPs/LIPUS treatment on ROS production in chondrocytes, cells were pretreated with 1 μg/mL LPS and handled with various concentrations (30 μg/mL, 60 μg/mL and 120 μg/mL) of PBNPs as well as various acoustic intensities (30 mW/cm 2 , 45 mW/cm 2 and 60 mW/cm 2 ) of LIPUS for 7 d. ROS production was estimated by using a uorescent probe DCFH-DA. As shown in Fig. 2A, the ROS production in cells treated with LPS was much higher than that in the control, and PBNPs reduced the LPS-induced ROS production in a dose-dependent manner. In addition, LIPUS treatment also alleviated LPS-induced ROS as the intensity increased (Fig. 2B). To further determine the effects of PBNPs/LIPUS combined treatment on LPS-induced ROS in chondrocytes, we treated cells with 120 μg/mL of PBNPs, 60 mW/cm 2 of LIPUS and a combination of both in severally. As a result, an evident reduction of ROS was observed in cells of the combination treatment group when compared with the PBNPs and the LIPUS group (Fig. 2C). These data indicated that the combined treatment could eliminate LPS-induced ROS better than the treatment of either PBNPs or LIPUS.

Effects of PBNPs/LIPUS treatment on apoptosis induced by LPS in chondrocytes
The effects of PBNPs/LIPUS treatment on apoptosis in chondrocytes were examined with ow cytometry assay and TUNEL staining. A substantial increase of apoptosis in the cells treated with LPS was observed compared with the control, yet PBNPs dose-dependently reduced apoptosis of chondrocytes induced by LPS ( Fig. 3A and 3B). Similarly, LIPUS treatment alleviated the apoptosis induced by LPS in reverse trends of increasing acoustic intensity ( Fig. 3C and 3D). Next, we combined PBNPs with LIPUS and found that the PBNPs/LIPUS combination treatment inhibited the apoptosis induced by LPS more effectively than either PBNPs or LIPUS alone ( Fig. 4A and 4B). We further detected the expression of the apoptosis-related proteins by using western blot. As shown in Fig. 4C, Bax, and cleaved caspase-3, which played a critical role in the regulation of cell apoptosis, were notably up-regulated in cells treated with LPS compared with the control. A radical reduction of Bax and cleaved caspase-3 was observed in the groups of PBNPs, LIPUS, and the combination treatment when compared with the LPS group. The protein expression of the combination treatment group was signi cantly reduced when compared with either the PBNPs or the LIPUS. Correspondingly, the antiapoptotic protein Bcl2 was signally reduced in the LPS group when compared with the control group. All the therapies with PBNPs, LIPUS, and the combination of both revealed an increase of Bcl2 expression compared with the LPS group, whereas the combined treatment promoted Bcl2 expression more e ciently in contrast to the single administration of either PBNPs or LIPUS. The ndings indicated that the combined treatment could alleviate LPS-induced apoptosis better than the single treatment of either PBNPs or LIPUS.

PBNPs/LIPUS treatment initiated the PI3K-Akt-mTOR pathway to reduce the LPS-induced ROS and apoptosis in chondrocytes
To determine the mechanism of PBNPs/LIPUS treatment reducing the LPS-induced ROS and apoptosis in chondrocytes, the protein expressions of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR were severally detected by western blot. Compared with the control, the protein levels of p-PI3K, p-Akt, p-mTOR revealed an intense diminish in the LPS-treated chondrocytes. However, combined treatment signi cantly increased the expression of p-PI3K, p-Akt, and p-mTOR compared with the LPS group (Fig. 5A), which indicated that PBNPs/LIPUS combined treatment initiated the PI3K-Akt-mTOR pathway. To further determine whether the PI3K-Akt-mTOR pathway mediates the effects of PBNPs/LIPUS combined treatment on ROS and apoptosis in chondrocytes, we pretreated cells with 100 nmol/L of wortmanin, and an inhibitor of PI3K. As a result has shown, wortmanin noticeably reduced the levels of p-PI3K, p-Akt, and p-mTOR in the cells with combined treatment (Fig. 5B). Additionally, PI3K inhibition sharply increased the ROS that was originally reduced by the combination therapy (Fig. 5C). Furthermore, a considerable increase in apoptosis was observed through ow cytometry assay and TUNEL staining after PI3K inhibition ( Fig. 5D and 5E). Also, the expression of Bax and cleaved caspase-3 in the combined treatment group were signi cantly increased after a wortmanin addition, while Bcl2 was notably reduced (Fig. 5F). These data indicated that PBNPs/LIPUS combination treatment reduced the LPS-induced ROS and apoptosis in chondrocytes by initiating the PI3K-Akt-mTOR pathway.
PBNPs/LIPUS treatment inhibited IL-1β and MMPs while activated JINK/c-Jun signal pathway in LPSincubated chondrocytes As results have shown, the IL-β, MMP3, and MMP13 protein expressions were dramatically elevated in LPS-incubated cells when compared with the control, whereas a prominent reduction was displayed in the groups of PBNPs, LIPUS, and the combined treatment when compared with the LPS group. There was also an evident decrease in the PBNPs/LIPUS combined treatment in contrast to either the PBNPs or LIPUS (Fig. 6A and 6B). These data revealed that PBNPs/LIPUS combined treatment could directly inhibit the in ammation and degeneration of ECM in chondrocytes. Furthermore, the p-JINK, JINK, p-c-Jun (ser308), and c-Jun protein expressions were detected. Compared with the control, expressions of p-JINK and p-c-Jun protein were signi cantly increased in LPS-treated cells. The protein expressions were largely reduced in the PBNPs/LIPUS combined treatment group in contrast to the LPS group ( Fig. 6C and 6D). It was, therefore, suggested that combined PBNPs/LIPUS therapy might diminish the in ammation and MMPs in LPS induced chondrocytes through the JINK/c-Jun signal pathway.
Effects of LIPUS/PBNPs treatment on article cartilage in rabbits with KOA Six weeks after the intervention, macroscopic observation of the femoral condylar cartilage samples in the ve groups was exhibited in Fig. 7A. In the KOA, severe hypertrophy, whiteness, and softness of articular cartilage with subchondral bone exposure were shown. Moreover, moderate hypertrophy, whiteness, and softness of articular cartilage were observed both in the KOA+PBNPs group and the KOA+LIPUS group. In the KOA+PBNPs+LIPUS group, the surface of articular cartilage was slightly pale and soft. Cartilage lesion grades measured by microscopic examination were shown in Table 3. Then the cartilage specimen was stained with hematoxylin and Safranin O was then examined under a microscope. The observations were shown in Fig. 7B and 7C. We found the articular cartilage surface was smooth and evenly stained with chondrocytes arranged in normal order in the control. Oppositely, the signi cant articular cartilage ssures, the apparent loss of staining extending from the surface to the depth as well as the reduction of the number of chondrocytes were observed in the KOA group. In the KOA+PBNPs and the KOA+LIPUS group, we observed moderate surface cracks in the articular cartilage with a medium loss of staining and chondrocytes. We then found minor surface cracks in the articular cartilage with mild loss of staining in the KOA+PBNPs+LIPUS group, and the chondrocytes showed slightly abnormal arrangement with decreased super cial cells. The Mankin scores after 6 wk of treatment were summarized in Table 4. As compared with the control group, the total scores were increased in the KOA. Additionally, both total scores in the KOA+PBNPs and the KOA+LIPUS were moderately lower than that in the KOA; however, scores of the KOA with combination treatment was lower than that in the KOA+PBNPs and the KOA+LIPUS respectively. Speci cally, when compared with the KOA, the PBNPs treatment mildly reduced the scores in subgroups 1 and 2, and the LIPUS treatment slightly reduced the scores in subgroups 2 and 3. Moreover, the combination treatment signi cantly decreased the scores in subgroup 1, 2, and 3 in contrast with the KOA.
Additionally, protein expressions of articular cartilage were detected by western blot (Fig. 8A). There was a moderate decrease of Bax, cleaved caspase-3, IL-1β, MMP3, and MMP13 and a moderate increase of Bcl2 after PBNPs treatment and LIPUS treatment. Furthermore, the regulation of combination treatment on these proteins manifested more apparently. These data indicated that PBNPs/LIPUS combined treatment e ciently inhibited the apoptosis of chondrocytes and the ECM damage in knee articular cartilage. Consistent with the result in vitro, PBNPs/LIPUS combined treatment activated the PI3K/Akt/mTOR signal pathway and suppressed the JINK/c-JUN axis as well (Fig. 8B).

Discussion
Currently, neither surgical nor non-surgical option can reverse the progress of OA on clinical treatment. In this study, we found that the protective effect of PBNPs/LIPUS combined treatment on KOA manifested as suppressing ROS and apoptosis in chondrocytes, which was mediated by PI3K/Akt/mTOR pathway initiation. Besides, the combined treatment also reduced in ammation and expression of MMPs through the suppressed JINK/c-Jun axis. This study was a novel description, and the results partly provided fundamental evidence for the clinical application of PBNPs/LIPUS combination therapy.
Chondrocyte apoptosis was correlated with the severity of cartilage degradation in OA [37]. Despite moderate levels of ROS played an important role in regulating normal chondrocytic activities including cell activation, proliferation, and matrix remodeling [7], a substantial number of researches suggested that ROS was the major cause of OA development [5,[38][39][40]. Excessive ROS could elicit protein oxidation, lipid peroxidation, and DNA damage resulting in cell damage and apoptosis [41][42][43]. This study demonstrated that all therapies with LIPUS, PBNPs, and combined PBNPs/LIPUS inhibited ROS in the LPS-treated chondrocytes and decreased apoptosis in vitro and vivo by up-regulating Bax and cleaved caspase-3 while down-regulating Bcl-2. It has been demonstrated that both PBNPs and LIPUS could scavenge ROS in cellular [20,44]. Consistent with previous studies, PBNPs and LIPUS reduced the ROS of LPS-pretreated chondrocytes in a dose-dependent and acoustic intensity-dependent manner. Of note, it was initially found that PBNPs (120 µg/mL) and LIPUS (60 mW/cm 2 ) could alleviate the apoptosis in chondrocytes and cartilage tissue. The anti-apoptotic effects of PBNPs have been rarely reported. Hollow Prussian blue nanozymes (HPBZs) possessed multienzyme activity assisted with Bi3 + and exerted antiapoptotic effects to protect cells from ischemia-induced injury by regulating the expression of anti-and pro-apoptotic proteins in vitro and vivo in a recent study [45]. Although the material structure of HPBZs did a little differ from PBNPs, the anti-apoptotic effects may be closely related to ROS scavenging. Nevertheless, the effects of LIPUS on apoptosis are controversial. Focused low-intensity pulsed ultrasound (FLIPUS) affected ECM production in KOA rabbits by decreasing chondrocyte apoptosis [46]. Conversely, LIPUS treatment promoted apoptosis and decreased the viability of endothelial cells, osteoclasts, and preadipocytes in human [47][48][49]. The opposite results may attribute to different parameters of LIPUS and different cells. We also found that brosis, matrix distribution, cartilage loss, and chondrocyte colonization were improved after the PBNPs/LIPUS treatment through macroscopic observation, HE, and Safranin O staining semi quantitated by using cartilage lesion grades and Mankin scores. Excitedly, the PBNPs/LIPUS combination treatment functioned as the most effective one in blocking apoptosis and ROS, thereby promoting cartilage damage recovery. In previous researches, LIPUS has been indicated a role in inducing greater internalization of nanomaterials into cells instead of affecting their viability [50,51]. Therefore, PBNPs entered cells more easily assisted with the mechanical function of LIPUS, achieving more powerful anti-apoptotic and ROS clearance effects.
The activation of the PI3K/Akt/mTOR signal pathway is widely recognized as a protective factor in OA. Ginsenoside Rg1 protected chondrocytes from IL-1β-induced mitochondria-activated apoptosis through the PI3K/Akt pathway [33]. The Akt activity enhancement by morroniside could be bene cial to chondrocyte survival [52]. Consistently, we found that PBNPs/LIPUS combination treatment activated PI3K/Akt/mTOR pathway in vitro and vivo, and PI3K inhibition by using wortmannin reduced the antiapoptotic and ROS clearance capability of the combination treatment.
It is believed that IL-1β and MMPs could act as possible markers of OA [53,54]. IL-1β was considered as one of the pro-in ammatory cytokines and highly associated with in ammatory pain [55,56]. In the pathological process of OA, the up-regulation of MMPs induced by IL-1β especially MMP-1 and − 13, presented as a key event during the irreversible changes of cartilage matrix degradation, for the reason that the type II collagen degeneration and the matrix proteoglycan consequent were released from the cartilage [57,58]. In previous studies, LIPUS has been demonstrated to decrease MMP's level in OA [59,60]. In this study, we illustrated that in LPS-treated chondrocytes, protein levels of IL-1β, MMP3, and MMP13 were mildly reduced in the LIPUS treatment group as well as the PBNPs treatment group, while those were signi cantly decreased in the combination treatment group, both in vitro and vivo. It has been demonstrated that PBNPs could dramatically reduce the LPS-induced expressions of ALT, IL-6, and IL-8 in serum [20], consistent with the anti-in ammation of PBNPs shown in our study. p38 and JNK enzymes involved in the MAPK pathway played a crucial role in the high-level MMPs expression in arthritic joints because a tightly regulated signaling pathway cascade activation was initiated by in ammatory cytokines namely IL-1β [11,61]. Our results also showed that JINK/c-Jun were activated by LPS whereas suppressed by the PBNPs/LIPUS combination treatment. Therefore, we assumed that the PBNPs/LIPUS combined treatment might reduce the levels of MMP3 and MMP13 by inhibiting the MAPK pathway, yet its mechanism remains to be explored.

Conclusion
In conclusion, our present study showed that combined PBNPs/LIPUS treatment, which was superior to either PBNPs or LIPUS in articular cartilage protection, could alleviate ROS and apoptosis of chondrocytes by activating the PI3K/Akt/mTOR pathway, reduce in ammatory cytokines and inhibit ECM degradation by reducing MMPs expression. Further studies are required to determine the effects of PBNPs/LIPUS treatment in the clinic.

List Of Abbreviations
Not applicable.

Declarations
Ethics approval and consent to participate All experiments were approved by the Animal Management Rule of the Chinese Ministry of Health and the Chongqing Medical University Animal Ethics Committee.

Consent for publication
Not applicable.

Availability of data and material
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.         KOA+PBNPs group, &p<0.05 vs. KOA+LIPUS group.