Targeting mitochondrial drug SkQ1 inhibits the progression of post-traumatic osteoarthritis by inhibiting mitochondrial oxidative stress

Targeting mitochondrial drug SkQ1 inhibits the progression of post-traumatic osteoarthritis by inhibiting mitochondrial oxidative stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeting mitochondrial drug SkQ1 inhibits the progression of post-traumatic osteoarthritis by inhibiting mitochondrial oxidative stress Zhenya Zhi, pengcheng Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5276838/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Post-traumatic osteoarthritis (PTOA) is a subtype of osteoarthritis (OA). At present, there are no ideal drugs that can effectively prevent and treat PTOA.The current strateg for treating PTOA is to control symptoms and reduce pain. SkQ1 is novel mitochondria-targeted antioxidant which can eliminate excessive intracellular ROS and exhibit anti-inflammatory effects. In this study, we evaluate the therapeutic effect of SkQ1 on PTOA and the mechanisms involved. Our results showed that SkQ1 significantly alleviated articular cartilage degeneration of PTOA in acute and chronic phase of PTOA rat model through inhibiting the oxidative stress. SkQ1 not only decreased the production of reactive oxygen species (ROS), MDA and 8-OHdg, but also suppressed the decrease of mitonchondrial membrane potential in PTOA rat model. We further found that SkQ1 protected mitochondrial function by inhibiting the release of cytochrome C and the expression of mitochondrial-related apoptotic pathway factors such as Bax, Bak, cleaved-caspase-3 and cleaved-caspase-9, increasing the copy number of mitochondrial DNA. In conclusion, SkQ1 may maintain mitochondrial function and inhibit the progression of PTOA by reducing ROS levels, inhibiting oxidative damage and apoptosis. SkQ1 may be used as a potential treatment for PTOA in the future. Health sciences/Biomarkers/Predictive markers Health sciences/Pathogenesis/Inflammation/Acute inflammation Post-traumatic osteoarthritis cartilage SkQ1 ROS Apoptosis mitochondrial dysfunction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Osteoarthritis (OA) is a chronic progressive degenerative disease of the whole joint, involving articular cartilage, subchondral bone, ligaments, joint capsules, and synovium. PTOA is a subtype of OA, with an incidence of about 12% of all OA [1] . PTOA occurs secondary to traumatic joint injury, such as fracture or soft tissue injury (cartilage surface, ligament, tendon and meniscus injury). Currently, there is no ideal treatment to prevent the progression of the disease, and PTOA cannot be completely avoided even after surgical treatment [2, 3] . Although the incidence of PTOA is not high compared with primary OA, PTOA has an earlier age of onset and more severe symptoms [4] , and therefore leads to a greater socioeconomic burden [5] . The diagnosis of PTOA depends on X-ray imaging, but early diseases and subtle changes are not easily detected, so PTOA is often diagnosed in the middle and later stages, and the treatment methods are mostly to relieve symptoms, such as reducing inflammation and pain [6] . In patients after joint replacement in the later stages, patients with PTOA are at higher risk of infection, stiffness, and revision compared with primary OA [7, 8] . Therefore, new strategies for diagnosis and timely intervention in the early stage of PTOA are urgently needed. A large number of previous studies have also shown that reactive oxygen species (ROS) play a major role in the occurrence and development of PTOA [9–11] . Abnormal mechanical and chemical stress can cause mitochondrial dysfunction in chondrocytes, resulting in increased production of oxygen free radicals and oxidative damage of tissues [11, 12] . Mitochondria are the main source of ROS. The early changes of damaged chondrocytes are characterized by increased activity of mitochondrial electron transport chain (ETC) and increased ROS, while the production of superoxide dismutase in damaged chondrocytes is decreased. Oxygen free radicals and metabolites lead to mitochondrial dysfunction, and mitochondrial contents such as cytochrome C (Cyt-C) and apoptosis inducing factor (AIF) are released from mitochondria. Thus, caspase in the cytoplasm is activated and apoptosis is initiated [13, 14] , leading to the occurrence and development of OA. Studies have shown that improving mitochondrial dysfunction and antioxidant therapy can alleviate the progression of PTOA [15–18] . SkQ1 is a mitochondrial-targeted antioxidant [19] , which can be enriched in mitochondria and has strong in situ clearance of mitochondrial ROS [20, 21] . Previous studies have shown that SkQ1 has a protective effect on oxidative stress-induced damage in various animal disease models [22–25] . However, the therapeutic potential of SkQ1 in PTOA is still unclear. Based on the above theories, we selected and verified the therapeutic effect of SkQ1 for the treatment of PTOA. In this study, a rat PTOA model was established to investigate the therapeutic effect of SkQ1 on PTOA in rats.The aim of this study is to evaluate the effects of SkQ1 inhibitting the progression of post-traumatic osteoarthritis by maintaining mitochondrial function. 2. Materials and Methods 2.1. Animal Models A total of 90 rats were used for the experiment, 7-weeks-old male Sprague-Dawley and Specific Pathogen Free (SPF), weighing 200–300 g. OA was induced by medial meniscal tear (MMT) surgery of the knee and anesthesia was administered by 3% isoflurane inhalation. Oral cephalexin was then given 1h before surgery and 12h and 24h after surgery. The operation was performed by longitudinally incising the medial knee joint of the right hind limb, then cutting the muscle layer and connective tissue until the medial collateral ligament was exposed. The medial collateral ligament was transected and the medial meniscus was resected. The rats in the sham operation group underwent the same surgical procedure but did not undergo MMT. The management of experimental animals was carried out in strict accordance with the recommendations of the United States "Guidelines for the Management and Use of Laboratory Animals". And the experimental procedures were carried out in compliance with the recommendations of the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). All experimental protocols were approved by the Ethics Committee for Hospital Animal Experiments of the Third Hospital of Hebei Medical University, China. 2.2 Experimental design The rats were numbered individually and then divided into 6 groups using a random number table (n = 15) and received sham operation or MMT operation on day 7. The experimental group was given intra-articular injection of 50ul of SkQ1 (MCE) aqueous solution with a concentration of 500nmol/L once a day starting from the day after the operation. The rats in group 1 and group 4 were set as the control group, and the rats in group 1 and group 4 received sham operation. Groups 2 and 5 were set as the model group, which received MMT surgery and the same volume of distilled water was injected into the joint. Groups 3 and 6 were set as the experimental groups, and the rats in the experimental groups were treated with MMT and SkQ1. At 2 weeks after operation, the rats in groups 1–3 were killed. At 6 weeks after operation, the rats in groups 4–6 were killed. The knee tissues of 15 rats in each group were collected for pathological section and detection of ROS, mitochondrial function, oxidative damage index, mitochondrial DNA copy number, and Western blot of other biological factors. 2.3 Acquisition of chondrocytes The rats were killed by cervical dislocation and sterilized with 75% alcohol. Knee cartilage was obtained under sterile conditions. Carefully scrape off the soft tissue from the cartilage and break the cartilage into 1mm 3 sizes. The cartilage was rinsed with sterile phosphate buffered saline (PBS) in a test tube and centrifuged at 1000 rpm for 5min. The supernatant was then removed and trypsin containing ethylenediamine tetraacetic acid (EDTA) was mixed with the residue and digested for 60 min at 37°C through an oscillator at 80 rpm. The mixture was then rinsed with sterile PBS solution and centrifuged again to remove the supernatant. A 0.2% type Ⅱ collagenase solution was then added to the precipitateand swirled at 37°C at 80 rpm for 4h. The samples were centrifuged again and the supernatant was discarded. The precipitate was then mixed with DMEM/F-12 medium to form a suspension, filtered three times with a sterile stainless steel mesh, and centrifuged once more to remove the DMEM/F-12 medium. Finally, the residue was suspended in a complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The density of chondrocytes was adjusted to 1×10 6 cells /ml and was used for all experiments no later than the first generation of chondrocytes. 2.4 H&E Staining(HE),Safranin O and Fast Green staining༈SO༉ After paraffin embedding, section, deparaffinization and hydration, the cartilage tissue of rats in each group was stained with HE: the sections were stained in hematoxylin solution for 3 minutes, followed by differentiation, water washing, and blue return. Then the cartilage tissue was stained in eosin solution for 3 minutes, and sealed with neutral resin. Safranin O and fast green staining: After being stained with Weigert hematoxylin for 5 min, slightly washed, differentiated with 1% alcohol hydrochloride for 1s, rinsed with tap water for 5 min, and stained with fast green dye for 5 min, the excess staining solution was washed off with water until the cartilage was colorless under the microscope. After being stained with safranin O for 3min, the excess staining solution was poured off, and the slices were treated with gradient concentrations of absolute ethanol and xylene, and then closed with neutral gum. The morphology and structure of cartilage in each group were observed by optical microscope. Cartilage degeneration degree scoring criteria of OARSI pathology scoring system were used for scoring [26] . 2.5 ROS measurement An appropriate amount of cell suspension was added with 10µmol/L DCFH-DA (Beyotime, China) and incubated at 37 ° C for 15 to 20 min. The mixture was mixed every 3 to 5 min, and the cells without staining were set as negative cell control. Cells were washed 3 times with PBS to remove DCFH-DA that did not enter the cells. ROS was detected by fluorescence spectrophotometer. 2.6 Malondialdehyde (MDA) and 8-OhdG (8-hydroxy-deoxyguanosine) determination Appropriate cell suspensions were obtained according to Malondialdehyde (MDA) Colorimetric Assay Kit (Elabscience, China), 8-OHdG (8-Hydroxydeoxyguanosine) ELISA Kit (Sangon Biotech, China) manual for testing. 2.7 Western blot analysis Total cellular proteins were separated by radio immunoprecipitation assay (RIPA) lysis buffer with protease inhibitors (Solarbio, China). Protein concentration was quantified by BCATM Protein Assay kit (Solarbio, China). The Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membrane. After incubation in 5% milk for 1 h at room temperature, the membranes were incubated with the primary antibodies (Affinity. USA) against Bcl2, Bcl2-XL, Bax, Bak, Caspase-3, Cleaved-Caspase3, Caspase-9, Cleaved-Caspase9 overnight at 4°C. The membranes were then incubated with the secondary antibody (Affinity. USA) for 1 hour at room temperature. Positive signals were detected by electrochemiluminescence (ECL, Abbkine, China). Western blot analysis of intracellular Cyt-c and AIF was performed according to the preceding steps after intracellular mitochondrial isolation according to the instructions of the Cell mitochondrial isolation Kit (Biyuntian, China). 2.8 Assessment of mitochondrial membrane potential (Δψm) 0.5ml of the prepared cell suspension was taken and operated according to the instructions of JC-1 Mitochondrial Membrane Potential Assay Kit (Beyotime, China), JC-1 staining working solution was added, and the cells were incubated in an incubator (37 ° C, After incubation in 5% CO2 for 20 min, the cells were collected and washed twice with JC-1 staining buffer, and then the cells were resuspended in JC-1 staining buffer. The results of mitochondrial membrane potential (Δψm) were detected by fluorescence spectrophotometer. 2.9 Determination of mitochondrial DNA (mtDNA) content Mitochondrial DNA copy number was quantified by the real-time-PCR based method using a mitochondrial DNA copy number assay kit (MCN2; Detroit R&D, Detroit, MI, USA) as per the manufacturer’s instructions. Reactions were performed with 10 ng of DNA, and mitochondrial DNA copy numbers were normalized with nuclear DNA copy number using the 2 –ΔΔCT method [27] . 2.10 Statistical analysis Statistical significance was checked by one-way or two-way ANOVA analysis followed by a Tukey's post hoc test, where a p value of less than 0.05 was considered as significant. The process was done using Graphpad Prism 9.0 software. 3. Results 3.1 SkQ1 alleviated articular cartilage degeneration of PTOA In order to observe the protective effect of SkQ1 on cartilage, we compared the pathological sections between different groups. We found that no obvious cartilage degeneration was observed in the cartilage tissue of the sham operation group at 2 and 6 weeks after surgery (Figure. 1 A and 1 B, Figure 2 A and 2 B). Obvious articular surface roughness and chondrocyte death were observed in the knee cartilage of rats at 2 weeks after surgery (Figure. 1 C and 1 D). More obvious cartilage fractures and loss of cartilage matrix appeared in the knee cartilage of rats at 6 weeks after surgery (Figure. 2 C and 2 D). However, cartilage degeneration was relatively mild after SkQ1 treatment (Figure 1 E and 1 F, Figure 2 E and 2 F), that is, more proteoglycan was retained and less cartilage matrix was lost at 2 and 6 weeks after intra-articular injection of SkQ1 compared with PTOA rats, as confirmed by OARSI scores (Figure 1 G and Figure 2 G, P < 0.05). Figure 1: SkQ1 alleviated cartilage degeneration at 2 weeks; scale bar is 50um; A and B are HE and SO staining of sham group, respectively; C and D are HE and SO staining of PTOA group, respectively; E and F are HE and SO staining of SkQ1 treatment group, respectively; n=3. * p<0.05, **p<0.01 Figure 2: SkQ1 suppresses the progression of cartilage degeneration at 6 weeks, scale bar is 50um, A and B are HE and SO staining of the sham-operated group, C and D are HE and SO staining of the PTOA group, E and F are HE and SO staining of the SkQ1 treatment group, n=3. *p<0.05, ****p<0.0001 3.2 SkQ1 attenuated ROS generation and oxidative damage in PTOA PTOA rats produced significant levels of ROS compared to the sham group (Figure 3 A and 3 D). However, when PTOA rats were treated with SkQ1, we found that their ROS levels were reduced at both week 2 and week 6 after operation. This indicated that ROS levels were increased in PTOA rats, but SkQ1 could rescue this phenomenon. Then, we evaluated the oxidative damage of rat cartilage by measuring MDA and 8-OHdG content. The results showed that PTOA caused increased MDA (Figure 3 B and 3 E) and 8-OHdg (Figure 3 C and 3 F) levels, indicating oxidative damage of lipids and nucleic acids. At 2 and 6 weeks after SkQ1 treatment, the results showed this oxidative damage was reduced. These results indicated that SkQ1 could reduce the elevated level of ROS and alleviate oxidative damage to cartilage in PTOA rats. Figure 3 SkQ1 attenuated ROS generation and oxidative damage in rat PTOA. ROS (A and D), MDA (B and E), and 8-OHdG (C and F) were tested at 2 and 6 weeks after administration. Data are presented as mean ± SEM values and representative of 5 independent experiments × 3 repeat. ****p<0.0001 3.3 SkQ1 reduces mitochondrial oxidative stress in OA rats The main function of mitochondria is to provide energy to cells by producing ATP. The energy generated during mitochondrial respiration is stored in the mitochondrial inner membrane in the form of electrochemical potential, resulting in the asymmetric distribution of proton plasma on both sides of the mitochondrial inner membrane, forming the mitochondrial membrane potential (ΔΨm) [28] . Therefore, we examined mitochondrial membrane potential and mitochondrial DNA copy number to represent mitochondrial function and mitochondrial synthesis. We found that PTOA resulted in reduced mitochondrial membrane potential and mitochondrial DNA copy number production compared to the sham surgery group (Figure 4). However, SkQ1 can significantly reverse mitochondrial dysfunction, which is manifested by the increase of mitochondrial membrane potential and mitochondrial DNA copy number. This suggests that SkQ1 may have the potential to alleviate mitochondrial oxidative stress induced by PTOA. Figure 4 SkQ1 can improve mitochondrial function, inhibite the decrease of mitochondrial membrane potential and increase mitochondrial DNA copy number. ΔΨm (A and C) and mtDNA copy number (B and D) at 2 and 6 weeks after administration of SKQ1. Data are presented as mean ± SEM values and representative of 5 independent experiments × 3. ****p<0.0001. 3.4 SkQ1 reduced mitochondria-associated chondrocyte apoptosis We collected cartilage cells from rats and quantified Bcl2, Bcl2-XL and Bax, Bak and other apoptosis-related factors. All western blot results are shown in Figure 5-k and Figure 6-k. The results showed the significantly increased levels of Bax, Bak, Caspase-3, Cleaved-Caspase3, Caspase-9, Cleaved-Caspase-9 in PTOA rats were reduced after SkQ1 treatment at 2 and 6 weeks (Figure 5 C-H, Figure 6 C-H). In addition, SkQ1 up-regulated the expression level of Bcl2, Bcl2-XL (Figure 5 A-B, Figure 6 A-B). Therefore, we hypothesized that SKQ1 may act by inhibiting mitochondria-related apoptotic pathways. Then, Western blotting was performed to analyze the expression levels in the cytoplasm of the rat knee chondrocytes after mitochondria separation, and it was found that the levels of Cyt-C and AIF in the cytoplasm of the OA rat chondrocytes were significantly increased. SkQ1 did decrease the levels of Cyt-C (Figure 5-I, Figure 6-I) and AIF (Figure. 5-J, Figure. 6-J) in the cytoplasm of PTOA rat cartilage. The Bcl2/Bax ratio was significantly increased after SkQ1 treatment, indicating that SkQ1 reduced the expression levels of molecules involved in the mitochondrial apoptosis pathway in PTOA chondrocytes, suggesting that SkQ1 may reduce the apoptosis of chondrocytes and alleviate cartilage degeneration by reducing mitochondrial oxidative stress. Figure 5: SkQ1 reduced apoptosis of cells with mitochondrial apoptosis at 2 weeks, SkQ1 increasedthe antiapoptotic factors Bcl2 and Bcl2-XL in PTOA (A and B, K), SkQ1 reduced the pro-apoptotic factors Bax, Bak, Caspase-3, Cleaved-Caspase3, Caspase-9, Cleaved-Caspase9, Cyt-c and AIF (C-K) in PTOA. Data are presented as mean ± SEM values and representative of 5 independent experiments × 3. *p<0.001, **p<0.01, ***p<0.001. ****p<0.0001. Figure 6: SkQ1 reduced apoptosis of cells with mitochondrial apoptosis at 6 weeks, SkQ1 increased the antiapoptotic factors Bcl2 and Bcl2-XL in PTOA (A and B, K), SkQ1 reduced the pro-apoptotic factors Bax, Bak, Caspase-3, Cleaved-Caspase3, Caspase-9, Cleaved-Caspase9, Cyt-c and AIF (C-K) in PTOA. Data are presented as mean ± SEM values and representative of 5 independent experiments × 3. *p<0.05，**p<0.01，***p<0.001****p<0.0001。 4. Discussion PTOA is often secondary to joint injury, mostly in young patients with OA, with pain and dysfunction as the main symptoms. Although various treatment strategies have been tested and proposed, it is not always effective in preventing the development of PTOA [3] . Therefore, it is urgent to further understand the pathogenesis of OA. Studies have shown that PTOA is associated with apoptosis and necrosis after acute cartilage injury, which is mainly caused by mitochondrial dysfunction and redox imbalance [10, 15] . ROS is a collective term for free radicals with oxygen molecules, such as superoxide anion (O 2 − ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH − ), and nitric oxide (NO) and its derivatives [29] . ROS can be scavenged by a variety of forms of scavengers, which play a role in maintaining the intracellular redox environment. The major scavengers of ROS are substances that produce energy by using oxygen, such as carotenoids, vitamin E, vitamin C, paraoxonase (PON), NADPH ubiquinone oxidoreductase (NQO1), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) [30] . SkQ1 is a synthetic product with antioxidant properties. Compared with traditional antioxidants, SkQ1 has a mitochondrial targeting effect, and its effective dose is much lower than that of conventional antioxidants [31] . Mitochondria is the main source of endogenous ROS [32] , and SkQ1 can effectively reduce the production of ROS at a lower dose [33] , which may also have lower toxic side effects. However, the relationship between SkQ1 and PTOA is still unclear. Here, we established a PTOA rat model induced by MMT surgery. Compared with the sham-operated group, the ROS level in the PTOA group was significantly increased. But when PTOA rats were treated with SkQ1, we found a significant reduction in ROS levels. These results indicated that SkQ1 was capable of scavenging the ROS levels induced by PTOA. MDA and 8-OHdg as markers of oxidative damage of lipids and nucleic acids, were increased under PTOA condition. While SkQ1 rescued these phenomena by reducing production of ROS, indicating that the oxidative damage of the cells was mitigated. Mitochondria are considered to be the intracellular source of ROS in animal cells, and excess ROS causes the antioxidant system to be overwhelmed and oxidative stress [34] . Although mitochondria are protected by outer and inner membranes, 1–5% of electrons may leak out during oxidative phosphorylation (OXPHOS), which further reacts with oxygen leading to ROS formation. For example, superoxide radicals (O 2 − ) and subsequent hydroxyl radicals (OH − ) and peroxynitrite anions (ONOO − ) cause oxidative stress by affecting the nucleus and mtDNA, as well as all cellular components, so increased ROS production is associated with hypoxia, mutation induction, cell transformation, and death [35] 。In this study, we found that mitochondrial dysfunction occurred in PTOA, as verified by the reduction of mitochondrial membrane potential, which was alleviated by the application of SkQ1。 Chondrocytes are the only cell species in cartilage tissue, and the number of chondrocytes alive determines the degree of joint degeneration [36] . In PTOA, ROS-induced apoptosis is considered to be the main form of chondrocyte death, especially after joint injury, and ROS accumulation may be mainly caused by mitochondrial dysfunction [37] . ROS can also induce pro-apoptotic Bcl-2 family proteins, such as Bak and Bax, antagonise the anti-apoptotic proteins Bcl-XL and Bcl-2 in the mitochondrial outer membrane, eventually leading to changes in mitochondrial outer membrane permeability and subsequent apoptosis [38, 39] . We therefore hypothesized that PTOA occurs as a result of ROS accumulation leading to oxidation of cardiolipin (the hallmark phospholipid in the mitochondrial inner membrane) and mitochondrial depolarization and subsequent opening of Bax/Bak channels in the mitochondrial outer membrane, leading to mitochondrial outer membrane permeability. These processes lead to the release of pro-apoptotic mediators. For example, cytochrome C and apoptosis-inducing factor (AIF).AIF can translocalizes to the nucleus, and then causes chromatin condensation and DNA fragmentation to initiate caspase-independent apoptosis. While the cytochrome C interacts with caspase-9 to form apoptotic bodies. And subsequently, the automatic activation of caspase-9 initiates the caspase cascade by activating the caspases-3, leading to the apoptosis of chondrocytes and the development of OA. This hypothesis was verified by this study. In addition, we demonstrated that mitochondrial DNA copy number was reduced in OA and this phenomenon could be prevented by the application of reducing agents [28] . Similarly, we found that SkQ1 could increase mitochondrial copy number that was inhibited under PTOA conditions. Therefore, we hypothesized that the mechanism by which SkQ1 inhibits PTOA may be to reduce mitochondrial oxidative stress and rescue mitochondrial function, thereby reducing the release of pro-apoptotic factors in mitochondria and reducing the apoptosis of chondrocytes. Western blot results supported the idea that SkQ1 did indeed reduced the levels of related pro-apoptotic molecules. Unfortunately, the pathological results showed that the application of SkQ1 delayed but did not completely prevent the development of PTOA, suggesting that the occurrence of PTOA may not be solely attributable to oxidative stress. 5. Conclusions In conclusion, we found that SkQ1 had the ability to inhibite the progression of PTOA in rats at both the acute (2 weeks) and chronic (6 weeks) phase after trauma, by reducing ROS production and alleviating oxidative stress damage to cells. SkQ1 also reversed the mitochondrial dysfunction under PTOA condition by increasing mitochondrial membrane potential and mitochondrial copy number, and reduced the release of pro-apoptotic factors and the expression level of apoptosis-related molecules in mitochondria. Therefore, Our data showed that the mitochondrial targeting drug SkQ1 still has application potential in the treatment of PTOA and is an effective drug for the treatment of PTOA。 Declarations Declaration of competing interest Announcement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment This study was supported by the Hebei Provincial Central Guidance Fund for Local Science and Technology Development Foundation (grant number 216Z7708G) Data availability statement All data generated or analysed during this study are included in this article. Author Contribution Z.Z. wrote the main manuscript text. W.P. reviewed and revised the manuscript. All authors reviewed the manuscript. References Brown T D, Johnston R C, Saltzman C L , et al. Posttraumatic osteoarthritis: a first estimate of incidence, prevalence, and burden of disease[J]. 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Mitochondrial antioxidant SkQ1 decreases inflammation following hemorrhagic shock by protecting myocardial mitochondria[J]. Frontiers in physiology, 2022, 13:1047909. Munro D, Treberg J R. A radical shift in perspective: mitochondria as regulators of reactive oxygen species[J]. Journal of Experimental Biology, 2017, 220(7):1170-1180. Averbeck D, Rodriguez-Lafrasse C. Role of Mitochondria in Radiation Responses: Epigenetic, Metabolic, and Signaling Impacts[J]. International journal of molecular sciences, 2021, 22(20). Hwang H S, Kim H A. Chondrocyte Apoptosis in the Pathogenesis of Osteoarthritis[J]. 2015, 16(11):26035-26054. Early J O, Fagan L E, Curtis A M , et al. Mitochondria in Injury, Inflammation and Disease of Articular Skeletal Joints[J]. Frontiers in immunology, 2021, 12:695257. Shi Y, Nikulenkov F, Zawacka-Pankau J , et al. ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis[J]. Cell death and differentiation, 2014, 21(4):612-623. Yang H, Xie Y, Yang D , et al. Oxidative stress-induced apoptosis in granulosa cells involves JNK, p53 and Puma[J]. Oncotarget, 2017, 8(15):25310-25322. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5276838","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":376172096,"identity":"5f3240ec-9fef-46c6-ad24-13a8cc2128ab","order_by":0,"name":"Zhenya Zhi","email":"","orcid":"","institution":"Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhenya","middleName":"","lastName":"Zhi","suffix":""},{"id":376172097,"identity":"c5e41255-41fb-4339-a974-2a162f69ff42","order_by":1,"name":"pengcheng Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYPACGzl+9h4GZggngSgtacaSPWdI03I4ccONHCK1GBw/e/h1QQ1Qy823Bx8X5hxm4GfPMWD4uQOPljN5adYzjqUbz7ydl2w8c9thBsmeNwaMvWdwazE7kGNmzNtgLdt3O8dMmheoxeBGjgEzYxseLeffgLQwMzbcPGP+G6TFnqCWGznGj3kbnBUn3OAxYwbbIkFAi/2NN2bMM46BAjnHWHrmtnQeiTPPCg724tEi2Z9j/LmgBhSVZww/F26zluNvT9744CceLUDAJo3M4wERB/BqYGBg/kxAwSgYBaNgFIx0AACzilOUrVlN1QAAAABJRU5ErkJggg==","orcid":"","institution":"Hebei Medical University","correspondingAuthor":true,"prefix":"","firstName":"pengcheng","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-10-16 14:53:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5276838/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5276838/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68919378,"identity":"3708695d-8f27-4b05-bc15-5364dab1aa44","added_by":"auto","created_at":"2024-11-13 13:26:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":88686,"visible":true,"origin":"","legend":"\u003cp\u003eSkQ1 alleviated cartilage degeneration at 2 weeks; scale bar is 50um; A and B are HE and SO staining of sham group, respectively; C and D are HE and SO staining of PTOA group, respectively; E and F are HE and SO staining of SkQ1 treatment group, respectively;\u003c/p\u003e\n\u003cp\u003en=3. * p\u0026lt;0.05, **p\u0026lt;0.01\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5276838/v1/ac4273cf840cde7f87f20fdc.jpg"},{"id":68919379,"identity":"bd4be457-50a2-4e25-8356-ca71b26f7332","added_by":"auto","created_at":"2024-11-13 13:26:31","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":86668,"visible":true,"origin":"","legend":"\u003cp\u003eSkQ1 suppresses the progression of cartilage degeneration at 6 weeks, scale bar is 50um, A and B are HE and SO staining of the sham-operated group, C and D are HE and SO staining of the PTOA group, E and F are HE and SO staining of the SkQ1 treatment group, n=3. *p\u0026lt;0.05, ****p\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5276838/v1/f2cd67a1f0cf59735d198a61.jpg"},{"id":68919382,"identity":"01675e3b-7a11-42dd-b87d-5979b215ba22","added_by":"auto","created_at":"2024-11-13 13:26:32","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":51184,"visible":true,"origin":"","legend":"\u003cp\u003eSkQ1 attenuated ROS generation and oxidative damage in rat PTOA. ROS (A and D), MDA (B and E), and 8-OHdG (C and F) were tested at 2 and 6 weeks after administration. Data are presented as mean ± SEM values and representative of 5 independent experiments × 3 repeat. ****p\u0026lt;0.0001\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5276838/v1/3ceab67281507129512f55f4.jpg"},{"id":68919800,"identity":"c4bb5cd4-77eb-43f7-8921-c1df280e3ca2","added_by":"auto","created_at":"2024-11-13 13:34:32","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":35699,"visible":true,"origin":"","legend":"\u003cp\u003eSkQ1 can improve mitochondrial function, inhibite the decrease of mitochondrial membrane potential and increase mitochondrial DNA copy number. ΔΨm (A and C) and mtDNA copy number (B and D) at 2 and 6 weeks after administration of SKQ1. Data are presented as mean ± SEM values and representative of 5 independent experiments × 3. ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5276838/v1/b59c0d1a943df2b1bbe784c3.jpg"},{"id":68919804,"identity":"771dc9cf-aba2-452a-ad5d-e8d59e2aa79e","added_by":"auto","created_at":"2024-11-13 13:34:32","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":59811,"visible":true,"origin":"","legend":"\u003cp\u003eSkQ1 reduced apoptosis of cells with mitochondrial apoptosis at 2 weeks, SkQ1 increasedthe antiapoptotic factors Bcl2 and Bcl2-XL in PTOA (A and B, K), SkQ1 reduced the pro-apoptotic factors Bax, Bak, Caspase-3, Cleaved-Caspase3, Caspase-9, Cleaved-Caspase9, Cyt-c and AIF (C-K) in PTOA.Data are presented as mean ± SEM values and representative of 5 independent experiments × 3. *p\u0026lt;0.001, **p\u0026lt;0.01, ***p\u0026lt;0.001. ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5276838/v1/02754a4030bba24f0e95ceb7.jpg"},{"id":68919381,"identity":"2bdb2f88-6c24-4795-a321-31230a11a3a7","added_by":"auto","created_at":"2024-11-13 13:26:32","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":53656,"visible":true,"origin":"","legend":"\u003cp\u003eSkQ1 reduced apoptosis of cells with mitochondrial apoptosis at 6 weeks, SkQ1 increased the antiapoptotic factors Bcl2 and Bcl2-XL in PTOA (A and B, K), SkQ1 reduced the pro-apoptotic factors Bax, Bak, Caspase-3, Cleaved-Caspase3, Caspase-9, Cleaved-Caspase9, Cyt-c and AIF (C-K) in PTOA. Data are presented as mean ± SEM values and representative of 5 independent experiments × 3. *p\u0026lt;0.05，**p\u0026lt;0.01，***p\u0026lt;0.001****p\u0026lt;0.0001。\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5276838/v1/0375837f3e24d3bf6eb7208b.jpg"},{"id":69504166,"identity":"0dddff3a-9977-4b3b-8836-eac76c0d8391","added_by":"auto","created_at":"2024-11-21 06:08:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":864968,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5276838/v1/de57037e-c3ab-4855-9714-8f31f87b17df.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Targeting mitochondrial drug SkQ1 inhibits the progression of post-traumatic osteoarthritis by inhibiting mitochondrial oxidative stress","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOsteoarthritis (OA) is a chronic progressive degenerative disease of the whole joint, involving articular cartilage, subchondral bone, ligaments, joint capsules, and synovium. PTOA is a subtype of OA, with an incidence of about 12% of all OA \u003csup\u003e[1]\u003c/sup\u003e. PTOA occurs secondary to traumatic joint injury, such as fracture or soft tissue injury (cartilage surface, ligament, tendon and meniscus injury). Currently, there is no ideal treatment to prevent the progression of the disease, and PTOA cannot be completely avoided even after surgical treatment \u003csup\u003e[2, 3]\u003c/sup\u003e. Although the incidence of PTOA is not high compared with primary OA, PTOA has an earlier age of onset and more severe symptoms\u003csup\u003e[4]\u003c/sup\u003e, and therefore leads to a greater socioeconomic burden \u003csup\u003e[5]\u003c/sup\u003e. The diagnosis of PTOA depends on X-ray imaging, but early diseases and subtle changes are not easily detected, so PTOA is often diagnosed in the middle and later stages, and the treatment methods are mostly to relieve symptoms, such as reducing inflammation and pain \u003csup\u003e[6]\u003c/sup\u003e. In patients after joint replacement in the later stages, patients with PTOA are at higher risk of infection, stiffness, and revision compared with primary OA\u003csup\u003e[7, 8]\u003c/sup\u003e. Therefore, new strategies for diagnosis and timely intervention in the early stage of PTOA are urgently needed.\u003c/p\u003e \u003cp\u003eA large number of previous studies have also shown that reactive oxygen species (ROS) play a major role in the occurrence and development of PTOA \u003csup\u003e[9\u0026ndash;11]\u003c/sup\u003e. Abnormal mechanical and chemical stress can cause mitochondrial dysfunction in chondrocytes, resulting in increased production of oxygen free radicals and oxidative damage of tissues \u003csup\u003e[11, 12]\u003c/sup\u003e. Mitochondria are the main source of ROS. The early changes of damaged chondrocytes are characterized by increased activity of mitochondrial electron transport chain (ETC) and increased ROS, while the production of superoxide dismutase in damaged chondrocytes is decreased. Oxygen free radicals and metabolites lead to mitochondrial dysfunction, and mitochondrial contents such as cytochrome C (Cyt-C) and apoptosis inducing factor (AIF) are released from mitochondria. Thus, caspase in the cytoplasm is activated and apoptosis is initiated \u003csup\u003e[13, 14]\u003c/sup\u003e, leading to the occurrence and development of OA. Studies have shown that improving mitochondrial dysfunction and antioxidant therapy can alleviate the progression of PTOA \u003csup\u003e[15\u0026ndash;18]\u003c/sup\u003e. SkQ1 is a mitochondrial-targeted antioxidant \u003csup\u003e[19]\u003c/sup\u003e, which can be enriched in mitochondria and has strong in situ clearance of mitochondrial ROS \u003csup\u003e[20, 21]\u003c/sup\u003e. Previous studies have shown that SkQ1 has a protective effect on oxidative stress-induced damage in various animal disease models \u003csup\u003e[22\u0026ndash;25]\u003c/sup\u003e. However, the therapeutic potential of SkQ1 in PTOA is still unclear. Based on the above theories, we selected and verified the therapeutic effect of SkQ1 for the treatment of PTOA. In this study, a rat PTOA model was established to investigate the therapeutic effect of SkQ1 on PTOA in rats.The aim of this study is to evaluate the effects of SkQ1 inhibitting the progression of post-traumatic osteoarthritis by maintaining mitochondrial function.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animal Models\u003c/h2\u003e \u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eA total of 90 rats were used for the experiment, 7-weeks-old male Sprague-Dawley and Specific Pathogen Free (SPF), weighing 200\u0026ndash;300 g. OA was induced by medial meniscal tear (MMT) surgery of the knee and anesthesia was administered by 3% isoflurane inhalation. Oral cephalexin was then given 1h before surgery and 12h and 24h after surgery. The operation was performed by longitudinally incising the medial knee joint of the right hind limb, then cutting the muscle layer and connective tissue until the medial collateral ligament was exposed. The medial collateral ligament was transected and the medial meniscus was resected. The rats in the sham operation group underwent the same surgical procedure but did not undergo MMT. The management of experimental animals was carried out in strict accordance with the recommendations of the United States \"Guidelines for the Management and Use of Laboratory Animals\". And the experimental procedures were carried out in compliance with the recommendations of the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). All experimental protocols were approved by the Ethics Committee for Hospital Animal Experiments of the Third Hospital of Hebei Medical University, China.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental design\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe rats were numbered individually and then divided into 6 groups using a random number table (n\u0026thinsp;=\u0026thinsp;15) and received sham operation or MMT operation on day 7. The experimental group was given intra-articular injection of 50ul of SkQ1 (MCE) aqueous solution with a concentration of 500nmol/L once a day starting from the day after the operation. The rats in group 1 and group 4 were set as the control group, and the rats in group 1 and group 4 received sham operation. Groups 2 and 5 were set as the model group, which received MMT surgery and the same volume of distilled water was injected into the joint. Groups 3 and 6 were set as the experimental groups, and the rats in the experimental groups were treated with MMT and SkQ1. At 2 weeks after operation, the rats in groups 1\u0026ndash;3 were killed. At 6 weeks after operation, the rats in groups 4\u0026ndash;6 were killed. The knee tissues of 15 rats in each group were collected for pathological section and detection of ROS, mitochondrial function, oxidative damage index, mitochondrial DNA copy number, and Western blot of other biological factors.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Acquisition of chondrocytes\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe rats were killed by cervical dislocation and sterilized with 75% alcohol. Knee cartilage was obtained under sterile conditions. Carefully scrape off the soft tissue from the cartilage and break the cartilage into 1mm\u003csup\u003e3\u003c/sup\u003e sizes. The cartilage was rinsed with sterile phosphate buffered saline (PBS) in a test tube and centrifuged at 1000 rpm for 5min. The supernatant was then removed and trypsin containing ethylenediamine tetraacetic acid (EDTA) was mixed with the residue and digested for 60 min at 37\u0026deg;C through an oscillator at 80 rpm. The mixture was then rinsed with sterile PBS solution and centrifuged again to remove the supernatant. A 0.2% type Ⅱ collagenase solution was then added to the precipitateand swirled at 37\u0026deg;C at 80 rpm for 4h. The samples were centrifuged again and the supernatant was discarded. The precipitate was then mixed with DMEM/F-12 medium to form a suspension, filtered three times with a sterile stainless steel mesh, and centrifuged once more to remove the DMEM/F-12 medium. Finally, the residue was suspended in a complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The density of chondrocytes was adjusted to 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells /ml and was used for all experiments no later than the first generation of chondrocytes.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 H\u0026amp;E Staining(HE),Safranin O and Fast Green staining༈SO༉\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAfter paraffin embedding, section, deparaffinization and hydration, the cartilage tissue of rats in each group was stained with HE: the sections were stained in hematoxylin solution for 3 minutes, followed by differentiation, water washing, and blue return. Then the cartilage tissue was stained in eosin solution for 3 minutes, and sealed with neutral resin. Safranin O and fast green staining: After being stained with Weigert hematoxylin for 5 min, slightly washed, differentiated with 1% alcohol hydrochloride for 1s, rinsed with tap water for 5 min, and stained with fast green dye for 5 min, the excess staining solution was washed off with water until the cartilage was colorless under the microscope. After being stained with safranin O for 3min, the excess staining solution was poured off, and the slices were treated with gradient concentrations of absolute ethanol and xylene, and then closed with neutral gum. The morphology and structure of cartilage in each group were observed by optical microscope. Cartilage degeneration degree scoring criteria of OARSI pathology scoring system were used for scoring \u003csup\u003e[26]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 ROS measurement\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAn appropriate amount of cell suspension was added with 10\u0026micro;mol/L DCFH-DA (Beyotime, China) and incubated at 37 \u0026deg; C for 15 to 20 min. The mixture was mixed every 3 to 5 min, and the cells without staining were set as negative cell control. Cells were washed 3 times with PBS to remove DCFH-DA that did not enter the cells. ROS was detected by fluorescence spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Malondialdehyde (MDA) and 8-OhdG (8-hydroxy-deoxyguanosine) determination\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAppropriate cell suspensions were obtained according to Malondialdehyde (MDA) Colorimetric Assay Kit (Elabscience, China), 8-OHdG (8-Hydroxydeoxyguanosine) ELISA Kit (Sangon Biotech, China) manual for testing.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Western blot analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTotal cellular proteins were separated by radio immunoprecipitation assay (RIPA) lysis buffer with protease inhibitors (Solarbio, China). Protein concentration was quantified by BCATM Protein Assay kit (Solarbio, China). The Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membrane. After incubation in 5% milk for 1 h at room temperature, the membranes were incubated with the primary antibodies (Affinity. USA) against Bcl2, Bcl2-XL, Bax, Bak, Caspase-3, Cleaved-Caspase3, Caspase-9, Cleaved-Caspase9 overnight at 4\u0026deg;C. The membranes were then incubated with the secondary antibody (Affinity. USA) for 1 hour at room temperature. Positive signals were detected by electrochemiluminescence (ECL, Abbkine, China). Western blot analysis of intracellular Cyt-c and AIF was performed according to the preceding steps after intracellular mitochondrial isolation according to the instructions of the Cell mitochondrial isolation Kit (Biyuntian, China).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Assessment of mitochondrial membrane potential (Δψm)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e0.5ml of the prepared cell suspension was taken and operated according to the instructions of JC-1 Mitochondrial Membrane Potential Assay Kit (Beyotime, China), JC-1 staining working solution was added, and the cells were incubated in an incubator (37 \u0026deg; C, After incubation in 5% CO2 for 20 min, the cells were collected and washed twice with JC-1 staining buffer, and then the cells were resuspended in JC-1 staining buffer. The results of mitochondrial membrane potential (Δψm) were detected by fluorescence spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Determination of mitochondrial DNA (mtDNA) content\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMitochondrial DNA copy number was quantified by the real-time-PCR based method using a mitochondrial DNA copy number assay kit (MCN2; Detroit R\u0026amp;D, Detroit, MI, USA) as per the manufacturer\u0026rsquo;s instructions. Reactions were performed with 10 ng of DNA, and mitochondrial DNA copy numbers were normalized with nuclear DNA copy number using the 2\u003csup\u003e\u0026ndash;ΔΔCT\u003c/sup\u003e method\u003csup\u003e[27]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical significance was checked by one-way or two-way ANOVA analysis followed by a Tukey's post hoc test, where a p value of less than 0.05 was considered as significant. The process was done using Graphpad Prism 9.0 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 SkQ1 alleviated articular cartilage degeneration of PTOA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;In order to observe the protective effect of SkQ1 on cartilage, we compared the pathological sections between different groups. We found that no obvious cartilage degeneration was observed in the cartilage tissue of the sham operation group at 2 and 6 weeks after surgery (Figure. 1 A and 1 B, Figure 2 A and 2 B). Obvious articular surface roughness and chondrocyte death were observed in the knee cartilage of rats at 2 weeks after surgery (Figure. 1 C and 1 D). More obvious cartilage fractures and loss of cartilage matrix appeared in the knee cartilage of rats at 6 weeks after surgery (Figure. 2 C and 2 D). However, cartilage degeneration was relatively mild after SkQ1 treatment (Figure 1 E and 1 F, Figure 2 E and 2 F), that is, more proteoglycan was retained and less cartilage matrix was lost at 2 and 6 weeks after intra-articular injection of SkQ1 compared with PTOA rats, as confirmed by OARSI scores (Figure 1 G and Figure 2 G, P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eFigure 1: SkQ1 alleviated cartilage degeneration at 2 weeks; scale bar is 50um; A and B are HE and SO staining of sham group, respectively; C and D are HE and SO staining of PTOA group, respectively; E and F are HE and SO staining of SkQ1 treatment group, respectively;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003en=3. * p\u0026lt;0.05, **p\u0026lt;0.01\u003c/p\u003e\n\u003cp\u003eFigure 2: SkQ1 suppresses the progression of cartilage degeneration at 6 weeks, scale bar is 50um, A and B are HE and SO staining of the sham-operated group, C and D are HE and SO staining of the PTOA group, E and F are HE and SO staining of the SkQ1 treatment group, n=3. *p\u0026lt;0.05, ****p\u0026lt;0.0001\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 SkQ1 attenuated ROS generation and oxidative damage in PTOA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePTOA rats produced significant levels of ROS compared to the sham group (Figure 3 A and 3 D). However, when PTOA rats were treated with SkQ1, we found that their ROS levels were reduced at both week 2 and week 6 after operation. This indicated that ROS levels were increased in PTOA rats, but SkQ1 could rescue this phenomenon. Then, we evaluated the oxidative damage of rat cartilage by measuring MDA and 8-OHdG content. The results showed that PTOA caused increased MDA (Figure 3 B and 3 E) and 8-OHdg (Figure 3 C and 3 F) levels, indicating oxidative damage of lipids and nucleic acids. At 2 and 6 weeks after SkQ1 treatment, the results showed this oxidative damage was reduced. These results indicated that SkQ1 could reduce the elevated level of ROS and alleviate oxidative damage to cartilage in PTOA rats.\u003c/p\u003e\n\u003cp\u003eFigure 3 SkQ1 attenuated ROS generation and oxidative damage in rat PTOA. ROS (A and D), MDA (B and E), and 8-OHdG (C and F) were tested at 2 and 6 weeks after administration. Data are presented as mean \u0026plusmn; SEM values and representative of 5 independent experiments \u0026times; 3 repeat. ****p\u0026lt;0.0001\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 SkQ1 reduces mitochondrial oxidative stress in OA rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe main function of mitochondria is to provide energy to cells by producing ATP. The energy generated during mitochondrial respiration is stored in the mitochondrial inner membrane in the form of electrochemical potential, resulting in the asymmetric distribution of proton plasma on both sides of the mitochondrial inner membrane, forming the mitochondrial membrane potential (\u0026Delta;\u0026Psi;m)\u003csup\u003e[28]\u003c/sup\u003e. Therefore, we examined mitochondrial membrane potential and mitochondrial DNA copy number to represent mitochondrial function and mitochondrial synthesis. We found that PTOA resulted in reduced mitochondrial membrane potential and mitochondrial DNA copy number production compared to the sham surgery group (Figure 4). However,\u0026nbsp;SkQ1 can significantly reverse mitochondrial dysfunction, which is manifested by the increase of mitochondrial membrane potential and mitochondrial DNA copy number. This suggests that SkQ1 may have the potential to alleviate mitochondrial oxidative stress induced by PTOA.\u003c/p\u003e\n\u003cp\u003eFigure 4 SkQ1 can improve mitochondrial function, inhibite the decrease of mitochondrial membrane potential and increase mitochondrial DNA copy number. \u0026Delta;\u0026Psi;m (A and C) and mtDNA copy number (B and D) at 2 and 6 weeks after administration of SKQ1. Data are presented as mean \u0026plusmn; SEM values and representative of 5 independent experiments \u0026times; 3. ****p\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 SkQ1 reduced mitochondria-associated chondrocyte apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe collected cartilage cells from rats and quantified Bcl2, Bcl2-XL and Bax, Bak and other apoptosis-related factors. All western blot results are shown in Figure 5-k and Figure 6-k. The results showed the significantly increased levels of Bax, Bak, Caspase-3, Cleaved-Caspase3, Caspase-9, Cleaved-Caspase-9 in PTOA rats were reduced after SkQ1 treatment at 2 and 6 weeks (Figure 5 C-H, Figure 6 C-H). In addition, SkQ1 up-regulated the expression level of Bcl2, Bcl2-XL (Figure 5 A-B, Figure 6 A-B). Therefore, we hypothesized that SKQ1 may act by inhibiting mitochondria-related apoptotic pathways. Then, Western blotting was performed to analyze the expression levels in the cytoplasm of the rat knee chondrocytes after mitochondria separation, and it was found that the levels of Cyt-C and AIF in the cytoplasm of the OA rat chondrocytes were significantly increased. SkQ1 did decrease the levels of Cyt-C (Figure 5-I, Figure 6-I) and AIF (Figure. 5-J, Figure. 6-J) in the cytoplasm of PTOA rat cartilage. The Bcl2/Bax ratio was significantly increased after SkQ1 treatment, indicating that SkQ1 reduced the expression levels of molecules involved in the mitochondrial apoptosis pathway in PTOA chondrocytes, suggesting that SkQ1 may reduce the apoptosis of chondrocytes and alleviate cartilage degeneration by reducing mitochondrial oxidative stress.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 5: SkQ1 reduced apoptosis of cells with mitochondrial apoptosis at 2 weeks, SkQ1 increasedthe antiapoptotic factors Bcl2 and Bcl2-XL in PTOA (A and B, K), SkQ1 reduced the pro-apoptotic factors Bax, Bak, Caspase-3, Cleaved-Caspase3, Caspase-9, Cleaved-Caspase9, Cyt-c and AIF (C-K) in PTOA. Data are presented as mean \u0026plusmn; SEM values and representative of 5 independent experiments \u0026times; 3. *p\u0026lt;0.001, **p\u0026lt;0.01, ***p\u0026lt;0.001. ****p\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003eFigure 6: SkQ1 reduced apoptosis of cells with mitochondrial apoptosis at 6 weeks, SkQ1 increased the antiapoptotic factors Bcl2 and Bcl2-XL in PTOA (A and B, K), SkQ1 reduced the pro-apoptotic factors Bax, Bak, Caspase-3, Cleaved-Caspase3, Caspase-9, Cleaved-Caspase9, Cyt-c and AIF (C-K) in PTOA. Data are presented as mean \u0026plusmn; SEM values and representative of 5 independent experiments \u0026times; 3. *p\u0026lt;0.05，**p\u0026lt;0.01，***p\u0026lt;0.001****p\u0026lt;0.0001。\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePTOA is often secondary to joint injury, mostly in young patients with OA, with pain and dysfunction as the main symptoms. Although various treatment strategies have been tested and proposed, it is not always effective in preventing the development of PTOA\u003csup\u003e[3]\u003c/sup\u003e. Therefore, it is urgent to further understand the pathogenesis of OA. Studies have shown that PTOA is associated with apoptosis and necrosis after acute cartilage injury, which is mainly caused by mitochondrial dysfunction and redox imbalance \u003csup\u003e[10, 15]\u003c/sup\u003e. ROS is a collective term for free radicals with oxygen molecules, such as superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), hydroxyl radical (OH\u003csup\u003e\u0026minus;\u003c/sup\u003e), and nitric oxide (NO) and its derivatives \u003csup\u003e[29]\u003c/sup\u003e. ROS can be scavenged by a variety of forms of scavengers, which play a role in maintaining the intracellular redox environment. The major scavengers of ROS are substances that produce energy by using oxygen, such as carotenoids, vitamin E, vitamin C, paraoxonase (PON), NADPH ubiquinone oxidoreductase (NQO1), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) \u003csup\u003e[30]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSkQ1 is a synthetic product with antioxidant properties. Compared with traditional antioxidants, SkQ1 has a mitochondrial targeting effect, and its effective dose is much lower than that of conventional antioxidants\u003csup\u003e[31]\u003c/sup\u003e. Mitochondria is the main source of endogenous ROS\u003csup\u003e[32]\u003c/sup\u003e, and SkQ1 can effectively reduce the production of ROS at a lower dose\u003csup\u003e[33]\u003c/sup\u003e, which may also have lower toxic side effects. However, the relationship between SkQ1 and PTOA is still unclear. Here, we established a PTOA rat model induced by MMT surgery. Compared with the sham-operated group, the ROS level in the PTOA group was significantly increased. But when PTOA rats were treated with SkQ1, we found a significant reduction in ROS levels. These results indicated that SkQ1 was capable of scavenging the ROS levels induced by PTOA. MDA and 8-OHdg as markers of oxidative damage of lipids and nucleic acids, were increased under PTOA condition. While SkQ1 rescued these phenomena by reducing production of ROS, indicating that the oxidative damage of the cells was mitigated. Mitochondria are considered to be the intracellular source of ROS in animal cells, and excess ROS causes the antioxidant system to be overwhelmed and oxidative stress\u003csup\u003e[34]\u003c/sup\u003e. Although mitochondria are protected by outer and inner membranes, 1\u0026ndash;5% of electrons may leak out during oxidative phosphorylation (OXPHOS), which further reacts with oxygen leading to ROS formation. For example, superoxide radicals (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) and subsequent hydroxyl radicals (OH\u003csup\u003e\u0026minus;\u003c/sup\u003e) and peroxynitrite anions (ONOO\u003csup\u003e\u0026minus;\u003c/sup\u003e) cause oxidative stress by affecting the nucleus and mtDNA, as well as all cellular components, so increased ROS production is associated with hypoxia, mutation induction, cell transformation, and death\u003csup\u003e[35]\u003c/sup\u003e。In this study, we found that mitochondrial dysfunction occurred in PTOA, as verified by the reduction of mitochondrial membrane potential, which was alleviated by the application of SkQ1。\u003c/p\u003e \u003cp\u003eChondrocytes are the only cell species in cartilage tissue, and the number of chondrocytes alive determines the degree of joint degeneration\u003csup\u003e[36]\u003c/sup\u003e. In PTOA, ROS-induced apoptosis is considered to be the main form of chondrocyte death, especially after joint injury, and ROS accumulation may be mainly caused by mitochondrial dysfunction\u003csup\u003e[37]\u003c/sup\u003e. ROS can also induce pro-apoptotic Bcl-2 family proteins, such as Bak and Bax, antagonise the anti-apoptotic proteins Bcl-XL and Bcl-2 in the mitochondrial outer membrane, eventually leading to changes in mitochondrial outer membrane permeability and subsequent apoptosis\u003csup\u003e[38, 39]\u003c/sup\u003e. We therefore hypothesized that PTOA occurs as a result of ROS accumulation leading to oxidation of cardiolipin (the hallmark phospholipid in the mitochondrial inner membrane) and mitochondrial depolarization and subsequent opening of Bax/Bak channels in the mitochondrial outer membrane, leading to mitochondrial outer membrane permeability. These processes lead to the release of pro-apoptotic mediators. For example, cytochrome C and apoptosis-inducing factor (AIF).AIF can translocalizes to the nucleus, and then causes chromatin condensation and DNA fragmentation to initiate caspase-independent apoptosis. While the cytochrome C interacts with caspase-9 to form apoptotic bodies. And subsequently, the automatic activation of caspase-9 initiates the caspase cascade by activating the caspases-3, leading to the apoptosis of chondrocytes and the development of OA. This hypothesis was verified by this study. In addition, we demonstrated that mitochondrial DNA copy number was reduced in OA and this phenomenon could be prevented by the application of reducing agents\u003csup\u003e[28]\u003c/sup\u003e. Similarly, we found that SkQ1 could increase mitochondrial copy number that was inhibited under PTOA conditions. Therefore, we hypothesized that the mechanism by which SkQ1 inhibits PTOA may be to reduce mitochondrial oxidative stress and rescue mitochondrial function, thereby reducing the release of pro-apoptotic factors in mitochondria and reducing the apoptosis of chondrocytes. Western blot results supported the idea that SkQ1 did indeed reduced the levels of related pro-apoptotic molecules. Unfortunately, the pathological results showed that the application of SkQ1 delayed but did not completely prevent the development of PTOA, suggesting that the occurrence of PTOA may not be solely attributable to oxidative stress.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn conclusion, we found that SkQ1 had the ability to inhibite the progression of PTOA in rats at both the acute (2 weeks) and chronic (6 weeks) phase after trauma, by reducing ROS production and alleviating oxidative stress damage to cells. SkQ1 also reversed the mitochondrial dysfunction under PTOA condition by increasing mitochondrial membrane potential and mitochondrial copy number, and reduced the release of pro-apoptotic factors and the expression level of apoptosis-related molecules in mitochondria. Therefore, Our data showed that the mitochondrial targeting drug SkQ1 still has application potential in the treatment of PTOA and is an effective drug for the treatment of PTOA。\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest Announcement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. \u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Hebei Provincial Central Guidance Fund for Local Science and Technology Development Foundation (grant number 216Z7708G)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this article.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZ.Z. wrote the main manuscript text. W.P. reviewed and revised the manuscript. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBrown T D, Johnston R C, Saltzman C L\u003cem\u003e, et al.\u003c/em\u003e Posttraumatic osteoarthritis: a first estimate of incidence, prevalence, and burden of disease[J]. Journal of orthopaedic trauma, 2006, 20(10):739-744.\u003c/li\u003e\n\u003cli\u003eSalman L A, Ahmed G, Dakin S G\u003cem\u003e, et al.\u003c/em\u003e Osteoarthritis: a narrative review of molecular approaches to disease management[J]. Arthritis research \u0026amp; therapy, 2023, 25(1):27.\u003c/li\u003e\n\u003cli\u003eDilley J E, Bello M A, Roman N\u003cem\u003e, et al.\u003c/em\u003e Post-traumatic osteoarthritis: A review of pathogenic mechanisms and novel targets for mitigation[J]. 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Oncotarget, 2017, 8(15):25310-25322.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Post-traumatic osteoarthritis, cartilage, SkQ1, ROS, Apoptosis, mitochondrial dysfunction","lastPublishedDoi":"10.21203/rs.3.rs-5276838/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5276838/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePost-traumatic osteoarthritis (PTOA) is a subtype of osteoarthritis (OA). At present, there are no ideal drugs that can effectively prevent and treat PTOA.The current strateg for treating PTOA is to control symptoms and reduce pain. SkQ1 is novel mitochondria-targeted antioxidant which can eliminate excessive intracellular ROS and exhibit anti-inflammatory effects. In this study, we evaluate the therapeutic effect of SkQ1 on PTOA and the mechanisms involved. Our results showed that SkQ1 significantly alleviated articular cartilage degeneration of PTOA in acute and chronic phase of PTOA rat model through inhibiting the oxidative stress. SkQ1 not only decreased the production of reactive oxygen species (ROS), MDA and 8-OHdg, but also suppressed the decrease of mitonchondrial membrane potential in PTOA rat model. We further found that SkQ1 protected mitochondrial function by inhibiting the release of cytochrome C and the expression of mitochondrial-related apoptotic pathway factors such as Bax, Bak, cleaved-caspase-3 and cleaved-caspase-9, increasing the copy number of mitochondrial DNA. In conclusion, SkQ1 may maintain mitochondrial function and inhibit the progression of PTOA by reducing ROS levels, inhibiting oxidative damage and apoptosis. SkQ1 may be used as a potential treatment for PTOA in the future.\u003c/p\u003e","manuscriptTitle":"Targeting mitochondrial drug SkQ1 inhibits the progression of post-traumatic osteoarthritis by inhibiting mitochondrial oxidative stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-13 13:26:26","doi":"10.21203/rs.3.rs-5276838/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c4600f44-6cce-4131-ab13-a69d5c0e48e0","owner":[],"postedDate":"November 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":40046284,"name":"Health sciences/Biomarkers/Predictive markers"},{"id":40046285,"name":"Health sciences/Pathogenesis/Inflammation/Acute inflammation"}],"tags":[],"updatedAt":"2024-11-21T06:08:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-13 13:26:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5276838","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5276838","identity":"rs-5276838","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}