Chondroprotective Effects of Gubitong Recipe via Inhibiting Excessive Mitophagy of Chondrocytes

Objective Osteoarthritis (OA) is the most common degenerative joint disorder and a leading cause of disability. A previous randomized controlled trial has shown that Gubitong (GBT) recipe can improve OA-related symptoms and articular function without noticeable side effects. However, the underlying mechanisms remain unclear. This study aims to explore the therapeutic mechanisms of the GBT recipe for OA through in vivo and in vitro experiments. Methods Rats of the OA model were established by Hulth surgery and intervened with the GBT recipe and then were subjected to pathological assessment of the cartilage. Matrix metalloproteinase 13 (MMP-13) expression in cartilage tissues was assessed by immunohistochemical staining. Chondrocytes were isolated from sucking rats and stimulated with LPS to establish an in vitro model. After intervened by water extraction of the GBT recipe, the fluorescent signal of Mtphagy Dye and mitochondrial membrane potential (Δψm) were detected to determine the states of mitophagy and mitochondrial dynamics of chondrocytes in vitro, respectively. Western blot test was used to detect levels of proteins related to catabolism of the cartilage matrix, mitophagy, and PI3K/AKT pathway. Results In in vivo experiments, the GBT recipe can effectively inhibit the cartilage degeneration of chondrocytes in OA rats, as well as markedly suppress the expression of MMP-13. In vitro experiments on LPS-induced chondrocytes exhibited increase in mitochondrial depolarization and excessive mitophagy, and the GBT recipe can alleviate these changes. LPS-stimulated chondrocytes showed increases in MMP-13, PINK1, and Parkin in cell lysates and LC3II/LC3I ratio in the mitochondrial fraction, and the GBT recipe can inhibit these increases in a dose-dependent manner. Moreover, the GBT recipe can attenuate the abnormal activation of PI3K/AKT pathway induced by LPS. Conclusion The GBT recipe exhibits chondroprotective effects through inhibiting excessive mitophagy of chondrocytes, which may be associated with its inhibitory effect on the abnormal activation of PI3K/AKT pathway.


Introduction
Osteoarthritis (OA) is a common and disabling condition characterized by degeneration of articular cartilage [1]. With an aging population and increasing rates of obesity, OA is becoming more prevalent than in previous decades [2]. A study based on the National Health Interview Survey (NHIS) of the United States showed that 22.7% of the adult population had at least one joint affected by OA and the incidence increased to 49.6% for those beyond the age of 65 [3]. Patients with OA often have pain, morning stiffness, crepitus on joint motion, and even instability or physical disability of joint, which impair quality of life and lead to a considerable socioeconomic burden [4].
Articular cartilage consists of a rich extracellular matrix (ECM) with a sparse dispersion of chondrocytes [5]. Adult articular cartilage is devoid of both innervation and vascularization, and chondrocytes are responsible for the anabolism and catabolism balance of ECM. Injured chondrocytes in osteoarthritic cartilage manufacture less ECM than usual, contributing to the irreversible degenerative process of OA [6,7]. e anabolic and catabolic processes of chondrocytes are intimately linked to mitochondrial functions [8]. Mitophagy, a selective form of autophagy that removes damaged or excessive mitochondria, is essential in maintaining cellular energy homeostasis and function [9]. However, excessive mitophagy can result in excessive mitochondrial oxidative stress and mitochondrial functions decline, leading to chondrocytes degeneration and cartilage destruction, and finally to OA [10,11].
Current management for OA focuses on relieving pain and inflammation, alleviating cartilage degeneration, and improving articular function [12]. Commonly, the treatment of OA includes pharmacological therapies, nonpharmacological therapies, and joint replacement surgery [13][14][15]. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most commonly recommended drugs for OA [15]. However, long-term applications of NSAIDs in patients with OA are controversial due to the gastrointestinal and cardiovascular side effects [16,17]. ere are no specific and efficacious disease-modifying drugs for OA yet [14]. us, it is desirable to develop a more effective and safer drug.
Traditional Chinese medicine (TCM) has been used to treat a variety of medical conditions including OA in China for thousands of years. Recently, some randomized controlled trials (RCTs) have demonstrated the therapeutic effect and safety of herbal medicine in treating OA [16][17][18][19]. Gubitong (GBT) recipe, a TCM prescription consisting of eight herbs, has also shown its therapeutic potential for patients with OA in a previous RCT [19]. However, the underlying mechanisms remain unclear. is study aims to explore the therapeutic mechanisms of the GBT recipe for OA through in vivo and in vitro experiments.

Animals
Feeding. Seven-week-old male Sprague-Dawley (SD) rats were purchased from SPF (Beijing) Biotechnology Co., Ltd. e rats were raised in a clean-grade animal room with a temperature of 23 ± 2°C and a humidity of 50 ± 10% in the Experimental Animal Center of China-Japan Friendship Hospital. All animal experiment procedures were approved by the Animal Care and Welfare Committee of China-Japan Friendship Hospital (No. zryhyy21-21-05-09). After 7 days of adaptive feeding, the rats were randomly divided into four groups, including blank control group, model group, GBT recipe group, and glucosamine sulfate group, according to the random digital table method.

OA Model Preparation and Intervention.
e rats were anesthetized by isoflurane inhalation. After fur was shaven off, the skins of the left keen joint were sterilized with iodophor. A surgical knife was used to transversely incise the skin of the left keen joint and patellar ligament. After the medial collateral ligament was severed and meniscus was drawn, anterior and posterior cruciate ligaments were severed. e incision was sutured layer by layer after a drawer test was positive. Penicillin (200,000 units) was injected into each rat that underwent surgery for successive three days to avoid infection. A typical daily dose of GBT recipe and glucosamine sulfate in the treatment of OA was 190 g and 1440 mg, and a human reference weight of an adult is 60 kg. us, the dose of GBT recipe and glucosamine to be administered is 3.2 g/kg and 1.7 mg/kg for an adult. According to the guide for dose conversion between animals and humans, the dose for rats is 6.3 times that of humans [20]. Based on this, the dose of GBT recipe in rats should roughly be 20.2 g/kg/day, and the dose of glucosamine sulfate should roughly be 150 mg/kg/d. Beginning on the 4th day following surgery, glucosamine sulfate and GBT recipe were given to rats once a day. Rats were sacrificed by inhalation of CO 2 (compressed CO 2 gas cylinder) at 4 weeks after administration. e left knee joint was separated for pathological assessment.

Pathological Assessment.
e knee joints of rats were fixed with 4% paraformaldehyde for 72 hours and decalcified in 10% ethylenediaminetetraacetic acid-Na 2 (EDTA-Na 2 ) for 6 weeks. e tissues were dehydrated, embedded, and sliced into 4 μm sections. e sections were then stained with hematoxylin and eosin (HE) and safranin O-fast green (SCO) staining for histological examination. Mankin's scoring method was used to evaluate the articular cartilage degeneration [21]. Each section was assessed by a welltrained researcher without being informed of the grouping.

Immunohistochemistry.
e paraffin-embedded tissues were used for the immunohistochemical analysis of MMP-13 expression in the articular cartilage of rats. In brief, after the slides were incubated with a blocking serum for 30 min, they were blotted and then overlaid with the primary antibody against MMP-13 for 2 h at room temperature. Subsequently, biotinylated secondary antibodies were added into the sections, followed by a peroxidase-labeled streptavidin-biotin staining technique. Finally, the samples were observed under a light microscope. e images from the immunohistochemistry samples were quantified using Image-Pro Plus software.

Isolation and Culture of Chondrocytes.
Five-day sucking rats were sacrificed by inhalation of CO 2 (compressed CO 2 gas cylinder). e knee joint cartilage was isolated in a sterile environment and digested using 0.25% trypsin for 1 hour. e cartilage was neutralized with DMEM containing 10% FBS and washed three times with PBS. After digested with 2 mg/ml type II collagenase for 6 hours, the joint cartilage was filtered with a 100-mesh stainless steel screen to prepare for chondrocytes suspensions. e suspensions were centrifuged to collect chondrocytes. Chondrocytes were seeded in a DMEM medium with 10% FBS, 50 U/ml penicillin, and 50 μg/ml streptomycin and cultured on polylysine-coated dishes. A second or third generation of chondrocytes was used for cell experiments.
2.9. MTS Assay. MTS assay was used to evaluate the effect of the GBT recipe on the vitality of chondrocytes. 1 × 10 4 chondrocytes were seeded into 96-well culture plates. After cells were completely adherent, the culture medium was discarded and the cells were treated with different concentrations of the GBT recipe for 12 hours. en, the cells were incubated for 4 hours in a DMEM medium. Subsequently, 20 μL MTS reagent was added to the cells, followed by another 20 min of incubation. A microplate reader was used to detect absorbance at a wavelength of 490 nm.
2.11. Mitochondrial Isolation. Chondrocyte mitochondria were isolated using a mitochondrial extraction kit. Chondrocytes were homogenized using a Dounce-type glass homogenizer in precold lysis buffer and centrifuged at 1000 g for 10 minutes. e mitochondria were then pelleted by centrifuging the crude supernatant for 10 minutes at 12,000 g. Precipitates were resuspended in 0.5 mL wash buffer and centrifuged at 1000 g for 5 min. Mitochondria were ultimately separated from the supernatant by centrifugation at 12,000 g for 10 minutes at 4°C.

Reverse Transcription and Quantitative Real-Time PCR.
RNA was extracted from chondrocytes and SW1353 cells using the column-based HiPure Total RNA Mini Kit. en, RNA concentration and purity were measured. 1 ug RNA was reverse transcribed into cDNA using a reverse transcription system. e qRT-PCR was performed in a total volume of 20 μl, with 2 μl of cDNA, 10 μl of SYBR Green qPCR Mix, and 2 μM of the forward and reverse primers in each tube. e CT values of each sample were acquired after the end of the reaction.

Western Blot Analysis.
e samples were lysed by RIPA buffer with 1% PMSF and 1% phosphatase inhibitors on ice Evidence-Based Complementary and Alternative Medicine 3 for 30 min. After centrifugation at 10,000 g for 10 minutes at 4°C, the supernatant was collected. e bicinchoninic acid (BCA) method was used to quantify the protein content. Protein samples were boiled after the addition of loading buffer, and 40 μg of each protein sample was loaded onto the SDS-PAGE gels. e protein was separated by SDS-PAG electrophoresis (SDS-PAGE) and transferred to PVDF membrane (70 v, 55 min). PVDF membrane was then blocked with 5% nonfat milk powder for 1 hour at room temperature.
e membranes were then incubated with primary antibodies overnight at 4°C. After washing with TBS containing 0.05% Tween-20 (TBST), membranes were incubated with secondary antibodies for 2 hours at room temperature. e protein samples were visualized using a chemiluminescence machine (Bio-Rad, USA). e primary antibodies associated parameters are listed in Table 1. 2.14. Immunofluorescence Staining. Chondrocytes were inoculated on a 48-well plate and cultured for 24 hours. e culture medium was discarded after completion and the plate was washed with PBS at 37°C. e cells were fixed for 15 minutes at room temperature with 4% paraformaldehyde and then permeabilized for 5 minutes with 0.1% Triton X-100/PBS. After washing three times with PBS, cells were blocked for 1 hour with an animal-free blocking solution. en, cells were incubated with antibodies against type II collagen (1 : 100 dilution) overnight at 4°C. Cells were washed three times with PBS and incubated with fluorescein (FITC)-conjugated goat anti-rabbit IgG (1 : 50 dilution) at room temperature for 1 hour. After the cells were washed three times with PBS, the nuclei were stained with DAPI and observed under an inverted fluorescence microscope.

Mitophagy Assay. A Mitophagy Detection Kit
(Dojindo Molecular Technologies) was used to detect mitophagy [22]. e chondrocytes were washed twice with DMEM and incubated with 100 nM Mtphagy Dye diluted in DMEM for 30 min at 37°C. After incubation, cells were washed twice with DMEM and continued to be incubated for another 1 h in the previous culture conditions. After mitochondrial staining, the dye was immobilized and fluorescence intensity varied according to pH value. In mitochondrial-lysosome fusion, Mtphagy Dye displayed higher fluorescence intensity, indicating mitophagy. e level of mitophagy was defined by the area of Mtphagy Dye per cell.

Flow Cytometry.
In the measurement of type II collagen, chondrocytes were fixed and incubated with type II collagen primary antibody (1 : 50 dilution) for 1 hour after discarding the culture supernatant. Subsequently, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibody for 30 minutes and then detected by flow cytometry. Mitochondrial membrane potential was measured using a mitochondrial membrane potential assay kit with JC-1 according to the manufacturer's protocol.

Statistical Analysis.
Statistical analysis was performed using SPSS 19 statistical package. Student's t-test was used to detect the statistically significant differences between experimental groups. Data were presented as mean ± standard deviation; a two-sided p < 0.05 was considered statistically significant.

Quality Control of GBT Recipe Extraction.
Representative components of each herb in the GBT recipe were selected according to the Chinese Pharmacopoeia (2020 edition) and relevant literature [23].

Protective Effects of GBT Recipe on Joints Cartilage of OA
Rats. Pathological changes in knee joint cartilage in each group are shown in Figure 2. Compared with the blank control group, the cartilage in the model group exhibited a successful establishment of the OA model and a significantly higher Mankin's score: the articular cartilage surface layer was fiberized, chondrocytes were severely proliferated and hypertrophied, cell lamination was completely disordered, and cytoplasmic vacuolization resulting from apoptotic cells was increased (Figures 2(a), 2(b), 2(e), and 2(f )). After GBT recipe treatment, cartilage lesion and matrix degradation were well ameliorated (Figures 2(c) and 2(g)). Meanwhile, the improvement of cartilage degeneration in the glucosamine sulfate group was inferior to that in the GBT recipe group (Figures 2(d) and 2(h)).

Effect of GBT Recipe on MMP-13 in the Articular Cartilage.
e results of immunohistochemistry showed that MMP-13 increased in cartilage of the OA model group (Figures 3(a)  and 3(b)). e GBT recipe and glucosamine sulfate could reduce the abnormal increase caused by OA, and the GBT recipe was more effective than glucosamine sulfate (p < 0.05) (Figures 3(c) and 3(d)).

Identification of Chondrocytes.
e identification of chondrocytes by type II collagen expression is shown in Figure 4.
e immunofluorescence analysis showed that nearly all chondrocytes were positive for type II collagen (Figures 4(a), 4(b), and 4(c)). e proportion of type II collagen-positive chondrocytes was further assessed by flow cytometry.
e results indicated that the proportion of chondrocytes expressing type II collagen was 98 ± 1.1%    (n � 3), and none for SW1353 cell-expressed type II collagen (Figures 4(d) and 4(e)). e qRT-PCR results demonstrated that chondrocytes expressed large amounts of type II collagen mRNA but the mRNA expression of type II collagen was not detectable in SW1353 cells (Figure 4(f )).   Evidence-Based Complementary and Alternative Medicine

Effects of GBT Recipe on LPS-Induced MMP-13 Expression in Chondrocytes. Western blot and RT-PCR analyses
showed that compared with LPS-free chondrocytes, LPSinduced chondrocytes had an increased expression of MMP-13 at the gene and protein levels. However, these changes in LPS-induced chondrocytes were reversed by the GBT recipe and DEX, and the GBT recipe showed a dose-dependent effect ( Figure 6).

Effects of GBT Recipe on LPS-Induced Mitophagy in
Chondrocytes. Mitophagy was detected using Mtphagy Dye. Compared with unstimulated chondrocytes, a significant increase in the fluorescent signal of Mtphagy Dye was detected in LPS-stimulated chondrocytes (Figures 7(a) and  7(b)). ese changes could be suppressed by 200 μg/ml GBT recipe and 100 nmol/ml DEX (Figures 7(c) and 7(d)).

Effects of GBT Recipe on LPS-Induced Mitochondrial Dynamics in Chondrocytes.
We used the JC-1 assay to measure mitochondrial membrane potential (Δψm) changes to see if LPS-induced mitophagy affects mitochondrial dynamics. LPS treatment significantly increased mitochondrial depolarization, as evidenced by a decrease in the red/green fluorescence intensity ratio (Figures 8(a) and 8(b)). 200 μg/ml GBTrecipe and 100 nmol/ml DEX stabilize the mitochondrial membrane potential (Δψm) significantly (Figures 8(c) and 8(d)). induced chondrocytes had an increased expression of PINK1 and Parkin. However, these changes in LPS-induced chondrocytes were reversed by the GBT recipe and DEX, and the GBT recipe showed a dose-dependent effect ( Figure 9).

Effects of GBT Recipe on PI3K/AKT Signaling Pathway in
Chondrocytes. Western blot revealed that the levels of PI3K and AKT proteins in LPS-induced chondrocytes were not changed compared with LPS-free chondrocytes, but the expression of p-PI3K and p-AKT was increased, suggesting that LPS abnormally activated the PI3K/AKTsignaling pathway in chondrocyte ( Figure 10). In addition, the phosphorylation levels of PI3K and AKT were reversed by the GBT recipe in a dose-dependent manner.

Discussion
Chondrocytes are the only cell type in cartilage and numerous studies have shown that OA often occurs with chondrocyte senescence and apoptosis [6,24,25]. During the pathologic process of OA, the capacity of chondrocytes to synthesize cartilage matrix was greatly reduced and large amounts of proteolytic enzymes were released, such as MMP-13, causing damage to the cartilage matrix [26]. us, improving the function of OA chondrocytes may be a target in treating OA [27].
Our study showed that the GBT recipe can effectively suppress the cartilage degeneration and apoptosis of the chondrocytes in the OA rat model. is was consistent with a previous study [28]. We found an increased expression of MMP-13 in the OA rat model by immunohistochemistry. MMP-13 was a hallmark of chondrocyte senescence and apoptosis and thus considered an important cause of the cartilage degeneration [29][30][31].
e intragastric administration of the GBT recipe can effectively reverse the high expression of MMP-13 in cartilage tissue of the OA rat model, which may indicate the therapeutic potential and mechanism of the GBT recipe for OA.
To further explore the therapeutic mechanisms of the GBT recipe for OA, we established an OA cell model by LPSinduced chondrocytes in vitro. e results showed that the mRNA and protein expressions of MMP-13 were significantly increased in LPS-induced chondrocytes and the GBT recipe can reverse this phenomenon in a dose-dependent manner.
is suggests that the GBT recipe can promote chondroprotection by inhibiting the expression of MMP-13 in inflammatory chondrocytes. JC-1 has been widely used to detect mitochondrial membrane potential as a fluorescent probe [32,33]. e JC-1 dye accumulates in mitochondria in a potential-dependent manner, which is usually indicated by a shift in fluorescence emission from green (535 nm) to red (595 nm). JC-1 forms aggregates with intense red fluorescence in healthy mitochondria with high ΔΨm, and monomers with green fluorescence when ΔΨm diminishes (depolarization) [34]. When the mitochondria are damaged, the mitochondrial membrane potential decrease in cells, the J-aggregate (red fluorescence) declines and the JC-1 monomer (green fluorescence) increases. e mitophagy detection kit is a common detection method for mitophagy after mitochondrial staining; the dye is immobilized and fluorescence intensity varies according to pH value [35,36]. In mitochondriallysosome fusion, Mtphagy Dye displays higher fluorescence intensity, indicating mitophagy.
Our study showed that there was a decrease in the red/ green fluorescence intensity ratio in LPS-induced chondrocytes, which indicated that the mitochondria membrane potential was depolarized and the mitochondrial function was impaired. Concurrently, the level of mitophagy in inflammatory chondrocytes was significantly elevated by analyzing the fluorescence intensity of Mtphagy Dye. ese results revealed that excessive mitophagy was induced by LPS stimulation causing mitochondrial dysfunction. In further chondrocyte experiments, we found that the expression of the following two key molecules in mitophagy regulation was much higher in the LPS group than in the control group: Parkin and PINK1, and the LC3II/LC3I ratio. ese results corroborate the excessive mitophagy in LPSinduced chondrocytes.
ROS are primarily generated in mitochondria, and mitochondrial abnormalities can induce ROS overproduction, leading to abnormal activation of the NFκB signaling pathway, eventually promoting MMPs expression [37][38][39][40]. e GBTrecipe and the positive control drug (Dex) can inhibit the mitochondrial depolarization and the expression of mitophagy-related proteins and thus alleviate excessive mitophagy. Overall, inhibiting the excessive mitophagy of inflammatory chondrocytes may be the key mechanism by which the GBT recipe protects chondrocytes.
Previous studies have demonstrated that the PI3K/AKT pathway is associated with the inflammatory response, apoptosis, and autophagy of chondrocytes and thus is closely related to the development of OA [41][42][43]. e inhibition of the PI3K/AKT pathway can reduce cartilage degeneration by regulating the expression of multiple downstream targets. Hence, the PI3K/AKT pathway is considered a potential therapeutic target in treating OA [44]. In our in vitro experiments, chondrocytes showed elevated levels of phosphorylated PI3K and phosphorylated AKT after LPS stimulation. Meanwhile, the GBT recipe reduced these changes in LPS-induced chondrocytes, suggesting that the GBT recipe can inhibit the abnormal activation of the PI3K/ AKT pathway, which may be associated with its chondroprotective effects and need further research in the future.
In conclusion, the GBTrecipe showed chondroprotective effects on OA rats and rat chondrocytes stimulated by LPS. e chondroprotective effects may be associated with the inhibition of excessive mitophagy and abnormal activation of the PI3K/AKT pathway. Taken together, we suggest that the GBT recipe can be an effective alternative therapy in preventing and treating OA. However, we did not explore the effect of the GBT recipe in synovitis and human OA chondrocytes in this experiment. More studies are still needed to further investigate and validate the protective effects of the GBT recipe for OA. Data Availability e data supporting the findings of this study are available from the corresponding author upon request.

Disclosure
Xin-bo Yu and Guang-yao Chen share the first authorship.

Conflicts of Interest
e authors declare that they have no conflicts of interest.