MicroRNA‐153‐3p increases autophagy in sevoflurane‐preconditioned mice to protect against ischaemic/reperfusion injury after knee arthroplasty

Abstract The use of tourniquet during total knee arthroplasty (TKA) can result in ischaemia/reperfusion injury (IRI). Of interest, microRNAs (miRs) are reported to be involved in various kinds of IRI due to their ability in modulating autophagy. Therefore, the study aimed to investigate the effect of miR‐153‐3p on autophagy in IRI in vitro and in vivo under sevoflurane preconditioning. In the in vitro model, chondrocytes from naive mice were treated with 0% FBS alone or in combination with sevoflurane. Additionally, in vivo assays were conducted in mouse models with tourniquet‐induced IRI after TKA under or without sevoflurane preconditioning. The pathological observation in vivo validated that sevoflurane preconditioning protected the knee joint against IRI. Moreover, miR‐153‐3p expression was diminished in chondrocytes of the in vitro model and in cartilage tissue of the in vivo model, but its expression was appreciably up‐regulated in the presence of sevoflurane preconditioning. Mechanistic study showed that miR‐153‐3p disrupted the interaction between Bcl‐2 and Beclin1 by targeting Bcl‐2, thereby facilitating autophagy in chondrocytes under sevoflurane preconditioning. Furthermore, the experiments in human chondrocytes also verified the protective effects of miR‐153‐3p against IRI were realized through inhibiting Bcl‐2. Collectively, miR‐153‐3p overexpression blocks the interaction between Bcl‐2 and Beclin1 via down‐regulation of Bcl‐2 to promote autophagy of chondrocytes, thus protecting knee joint against IRI after TKA under sevoflurane preconditioning.


| INTRODUC TI ON
The use of a tourniquet is employed in total knee arthroplasty (TKA) procedures in order to prevent blood loss. 1 Furthermore, in the event a tourniquet is inflated, ischaemia will be induced to an extremity, the release of which results in subsequent ischaemia/ reperfusion injury (IRI) to localized skeletal muscle, to systemic circulation and in some instances to vital distant organs such as the heart, kidneys, lungs and brain. 2 IRI of limbs and distal organs induced by tourniquet has been reported by various studies following TKA. 3 Similarly, accumulating studies continue to highlight IRI as an issue requiring further investigations in knee arthroplasty. 4-7 A previous study concluded that myocardial IRI could be repressed by means of sevoflurane-increased autophagy. 8 Sevoflurane has also been reported to attenuate IRI in certain organs including the heart, lung and liver in mice administered with sevoflurane, illustrated by a superior recovery following IRI. [9][10][11][12][13] Additionally, a recent study demonstrated that the use of hyperbaric oxygen preconditioning confers neuro-protective effects against IRI in neurocyte a process of which involves the induction of autophagy, highlighting the possible link between autophagy and protection against IRI following TKA. 14 The ability of microRNAs (miRs) to regulate gene expression at a post-transcriptional level has been shown to influence ischaemic preconditioning, ischaemic post-conditioning and IRI. 15 Additionally, miR-153-3p has been implicated in acute graft-versus-host disease. 16 Furthermore, studies have indicated that B-cell lymphoma-2 gene (Bcl-2) is directly targeted by miR-153-3p. 17 As a suppressor of apoptosis, Bcl-2 has been widely documented to promote cell survival and inhibits cell death. 18 Furthermore, it has been observed that in human SGC-7901 cells, Beclin1-dependent autophagy is stimulated by small interfering RNA-mediated Bcl-2 silencing. 19 Existing literature has revealed that the disrupted interaction between Beclin1 and Bcl-2 arises following activation of AMPK which enhances autophagy and decreases cardiomyocyte apoptosis, which has been shown to be beneficial for the treatment of diabetic cardiomyopathy. 20 Similarly, another study explored the contribution of the interaction between Beclin1 and Bcl-2 induced by mammalian Ste20-like kinase 1 (Mst1) and the suppression of autophagy. 21 Notably, Peng et al provided evidence indicating that myocardial tissue is protected against IRI following ischaemic preconditioning (IPC) which blocks the interaction of Beclin1 and Bcl-2 to increase autophagy. 22 Based on the aforementioned studies, we hypothesized that miR-153-3p promotes the protection and alleviates the effects of IRI by mediating autophagy through the interaction between Beclin1 and Bcl-2. Hence, the study was designed in order to investigate the effect of miR-153-3p on IRI following TKA as well as the underlying mechanism involved with Beclin1, Bcl-2 and autophagy.

| Ethics statements
This study was performed with the approval of the Animal Committee of Linyi People's Hospital. Extensive efforts were made to minimize the suffering of the animals during our study.

| Animals
A total of 66 male BL6/C57 mice (aged 7 weeks; weighing 21-25 g) at specific pathogen-free (SPF) grade were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Among the mice, 50 mice were subjected to TKA experiments, while the remaining mice were used to isolate primary chondrocytes.

| Isolation and identification of primary chondrocytes
The mice were killed, after which their bilateral knee cartilages were obtained under aseptic conditions. The cartilages were washed with phosphate-buffered saline (PBS) with the soft tissue as well as the bone tissues around the articular cartilages subsequently removed.
The cartilages were treated with 2.5 g/L trypsin (Sigma-Aldrich

| RT-qPCR
The total RNA was extracted from cartilage tissues and primary chondrocytes using a Trizol RNA kit. The concentration of the RNA was subsequently determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc). The RNA was then reversely transcribed into cDNA using the ReverTra Ace ® qPCR RT Master Mix with gDNA Remover. The primers of miR-153-3p, collagen ΙΙ, Bcl-2 and Beclin1 were designed and synthesized by Shanghai Sangon Biotechnology Co. Ltd., (Table 1), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 employed as the internal references. Fluorescence quantitative PCR was performed using the Bio-Rad CFX96 Touch™ system based on the provided instructions of the 2× RealStar Green Mixture Kit (GenStar). The 2 −ΔΔCt method was applied to calculate the relative expression of the target genes. The experiment was repeated 3 times.

| Animal treatment
Fifty mice were randomly selected to establish the IRI mouse model. After the mice had been killed, the bilateral femurs of mice were dissected, with 1 mm knee joint cartilage cut using a blade along the vertical direction of the long axis of the blade. The lateral section diameter of the femur was measured, after which a needle with a diameter of 0.2 mm was selected as a simple prosthesis.
The mice were anesthetized by intraperitoneal injection with 1% pentobarbital sodium (0.2 mL for each mice), fixed on an operating table and sterilized with conventional iodine and alcohol. The skin, subcutaneous tissue and joint capsule were cut in the middle of the knee to expose the knee joint. A longitudinal incision was then made to the joint capsule from the medial patella, with the right knee joint exposed by dislocating the patellar dislocation via knee flexion. An incision was made to the anterior cruciate ligament as well as the medial and lateral meniscus for tibial subluxation purposes. Next, osteotomy of tibia and femur was performed, after which bleeding was stopped using a tourniquet, with the pulp reamed accordingly. The model was then evaluated, with a simple prosthesis inserted into the joint after the prosthesis had been sterilized and disinfected under high-temperature conditions. The deep fascia and skin were sutured in a layer by layer manner, after which the tourniquet was gradually deflated. After the wound had been covered with alcohol gauze, 200 U of gentamicin was injected into the muscle of the mice for three days (twice per day) after operation. Prior to tourniquet deflation, the modelled mice were permitted to inhale 2.4% sevoflurane (0426, Fuso Pharmaceutical Industries Ltd.) in a continuous manner until 30 minutes after suture. [23][24][25] Among the sham-operated mice, the right knee joint capsule was opened and closed under identical conditions. A total of 41 mice were successfully modelled, with a model success rate of 82%.
Six sham-operated C57/BL6 mice were randomly selected and six IRI-modelled mice that did not inhale sevoflurane. Besides, 24 IRImodelled mice inhaled sevoflurane and either remained untreated or were injected with inhibitor negative control (NC) + overexpression (oe)-NC lentivirus, miR-153-3p inhibitor + oe-NC lentivirus or miR-153-3p inhibitor + oe-Bcl-2 lentivirus or not (n = 6). On the third day post-operation, the mice were punctured inward and backward in close proximity to the inferior pole of the patella via the lateral margin of the ligamentum patellae. After touching the prosthesis, lentiviruses were injected into the articular cavity with a dose of 1 × 10 6 (50 μL). After 14 d of routine feeding, the tissues were collected for further examination.

| Starvation and sevoflurane exposure of mouse primary chondrocytes
The primary chondrocytes were isolated from mice that had not been subjected to any operative procedures and cultured in a medium containing 10% FBS (control group). When cell confluence reached approximately 90%, the chondrocytes were passaged at a density of 2 × 10 5 cells/dishes. The medium was replaced by a FBS free medium. A portion of the cells were then incubated at 37°C with 5% CO 2 (0% FBS group), while the remaining portion of the cells were incubated at 37°C in a 5% CO 2 incubator with sevoflurane gas introduced using an anaesthetic vaporizer (Drager Medical AG) at a rate of 2 L/min until the concentration of sevoflurane eventually reached 1%-6% (0% FBS + sevoflurane group). The concentration of sevoflurane was monitored every minute using an anaesthesia analyser (Drager Medical AG) to ensure that the concentration was within the normal range. The chondrocytes were treated for 24 hours and used for subsequent experimentation. 26

| Human chondrocytes isolation and culture
Cartilage specimens collected from patients that had undergone operative procedures at our hospital were promptly washed with normal saline in order to remove any remaining blood and tissue fluids and stored at low-temperature sterile conditions. After 3 rinses with PBS containing 1% penicillin-streptomycin (100 U/mL penicillin and 100 μg/mL streptomycin), the degenerated cartilage tissue samples were minced into pieces of less than 1 mm 3 , followed by sequential detachment by 0.25% trypsin at 37°C for 30 minutes and by 0.2% collagenase II (prepared by DMEM containing 10% FBS and 1% penicillin-streptomycin) under shaking at 37°C for 16 hours. Subsequently, chondrocytes were subjected to centrifugation, cultured in 5 mL DMEM culture solution with 20% PBS and constructed into cell suspension. The cells were then seeded into a culture flask at a density of 2.5 × 10 5 cells/mL and cultured at 37°C in 5% CO 2 . After 48 hours, the culture medium was changed based on cell adherence. The growth rate and morphology of the cells were observed, recorded and photographed
using an inverted microscope on a daily basis. The culture medium was changed regularly every 3 days.

| Chondrocyte treatment
The primary chondrocytes from naive samples were seeded into a

| Haematoxylin-eosin staining
On the 14th day post-operation, the mice were killed, with the

| Co-localization of Bcl-2 and Beclin1
The chondrocytes were washed 3 times with PBS and fixed with 4% phosphonoformate (PFA) at room temperature for 30 minutes. The chondrocytes were washed 3 times with PBS, sealed with 1% bovine serum albumin (BSA) and incubated with primary antibodies (Abcam Inc) to anti-rabbit Bcl-2 and anti-rabbit Beclin1 at 4°C overnight.
The chondrocytes were then subjected to additional PBS washing, incubated with FITC-labelled mouse anti-rabbit immunoglobulin G (IgG) and CY3-conjugated goat antimouse IgG antibodies (Santa Cruz Biotechnology Inc) at room temperature for 1 hour, followed by staining with 4',6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich Chemical Company). The chondrocytes were mounted and observed under a fluorescence microscope.

| Western blot analysis
The

| Statistical analysis
Statistical analyses were conducted using SPSS 21.0 (IBM).
Measurement data were expressed as mean ± standard deviation.
If the data conformed to normal distribution and homogeneity of variances, the comparison between two groups in non-paired design was analysed via non-paired t test, and comparisons among multiple groups were performed by one-way analysis of variance (ANOVA), followed by Turkey's post hoc test. P < .05 was considered to be indicative of statistical significance.

| miR-153-3p is up-regulated in IRI chondrocytes and mice pre-treated with sevoflurane
The mouse primary chondrocytes were initially isolated and cultured, after which the matrix secreted by the chondrocyte was stained with Alcian blue in order to confirm the chondrocyte characteristics. RT-qPCR was conducted to determine collagen ΙΙ expression in the chondrocytes. As depicted in Figure 1A higher levels were identified in the IRI mice treated with sevoflurane (P < .05; Figure 1E). Taken together, the use of tourniquet led to IRI during TKA, which could be alleviated by sevoflurane preconditioning. Furthermore, the expression of miR-153-3p was elevated following sevoflurane preconditioning during TKA, highlighting the potential crucial role of miR-153-3p in protecting against IRI.

| miR-153-3p disrupts the interaction between Bcl-2 and Beclin1 by targeting Bcl-2 to promote chondrocyte autophagy in vitro
After the chondrocytes had been treated with miR-153-3p inhibitor in the presence of 0% FBS+ sevoflurane, the co-localization of Bcl-2 and Beclin1 was assessed accordingly. The results obtained indicated that following miR-153-3p inhibition, the co-localization areas of Bcl-2 and Beclin1 were both markedly increased ( Figure 3A).

Additionally, Co-IP depicted that inhibition of miR-153-3p increased
Bcl-2-bound Beclin1 protein ( Figure 3B-E). Western blot analysis was performed in order to determine the expression of Beclin1 and autophagy-related factors (P62, LC3-I and LC3-II). As depicted in Figure 3F, when miR-153-3p was inhibited in chondrocytes treated with 0% FBS+ sevoflurane, P62 and LC3-I expression was increased while the expression of LC3-II was decreased, indicating the decrease of autophagy. In conclusion, in regard to the protection mechanism of sevoflurane preconditioning, miR-153-3p silencing promotes the interaction between Bcl-2 and Beclin1 to inhibit chondrocyte autophagy.

| miR-153-3p alleviates IRI by promoting chondrocyte autophagy in vivo
The IRI mice pre-treated with sevoflurane were established, and

| D ISCUSS I ON
Ischaemic/reperfusion injury has been widely documented to not only inflict injury at the site of ischaemia, but can also lead to a cascade of damage to distal and non-ischaemic organs. 31 The use of tourniquet in TKA has been speculated to contribute to IRI in localized skeletal muscle, systemic circulation as well as vital distant organs. 2 The emerging role of autophagy in various cardiovascular diseases, including that of heart failure, dilated cardiomyopathy and ischaemic heart disease has been discussed in many reports. 32 Moreover, a previous study revealed the existence of a correlation between miRs with autophagy and upstream autophagy signalling pathways, including p53, AMPK and Bcl-2. 33     Furthermore, a key finding of the current study illustrated that miR-153-3p overexpression promotes autophagy by means of disrupting the interaction between Bcl-2 and Beclin1 in IRI mice following TKA. It is well acknowledged that Beclin1 is involved at various stages of myocardial IRI. 38 Tang et al concluded that HBV × protein increases autophagy by increasing Beclin1 expression. 39 Moreover, a previous study revealed promoted autophagy in N2a with IRI after melatonin treatment, accompanied by an elevation in the expressions of Beclin1 and LC3-II expression. 40 Bcl-2 has been proven to interact with Beclin1 and contribute to the suppression of Beclin1-dependent autophagy. 41 Du et al revealed that Bcl-2 silencing promotes Beclin1-dependent autophagy in gastric cancer cells. 19 Disruption of the interaction between Bcl-2 and Beclin1 has been implicated in maslinic acid-promoted autophagy in rat pheochromocytoma cells. 42 Another study indicated that autophagy is induced by blocking the interaction between Bcl-2 and Beclin1, as a result of BH3 mimetic S1 promoting autophagy via blockade of Bcl-2/Beclin1 interaction in human glioma cells. 43 More importantly, a previous investigation demonstrated that Bcl-2 was directly targeted and negatively regulated by miR-153-3p. 17 In conclusion, the findings of our study present in vivo and in vitro evidence supporting the notion that miR-153-3p alleviates IRI in sevoflurane pre-treated chondrocytes and mice following TKA, via the induction of autophagy by blocking the interaction between Bcl-2 and Beclin1 ( Figure 6). Our findings highlight the potential of miR-153-3p up-regulation as a potential clinically viable target capable of enhancing the protection of sevoflurane preconditioning against IRI following TKA. The potential role of sevoflurane in regulation of Beclin1 was not fully explored. Therefore, more detailed experiments are needed in the future to confirm the findings of our study.

ACK N OWLED G EM ENTS
We would like to acknowledge the reviewers for their helpful comments on this study.

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
Shuang Qiu, Benjuan Liu and Xueqin Wang designed the study. Lina Zhong and Xiao Han collated the data, Fuli Mi and Yanshuai Mo contributed to drafting the manuscript. All authors have read and approved the final submitted manuscript.

E TH I C A L A PPROVA L
This study was performed with the approval of the Animal Committee of Linyi People's Hospital. Extensive efforts were made to minimize the suffering of the animals during our study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.