The effects of intra-articular oxamate on anterior cruciate ligament transection-induced experimental osteoarthritic rats

The effects of intra-articular oxamate on anterior cruciate ligament transection-induced experimental osteoarthritic rats | 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 Research Article The effects of intra-articular oxamate on anterior cruciate ligament transection-induced experimental osteoarthritic rats Zhi-Hong Wen, Chun-Sung Sung, Sung-Chun Lin, Zhi-Kang Yao, Yu-Cheng Lai, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2540780/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 Background: Osteoarthritis (OA) is the most common form of joint disorder and arthritis worldwide. Joint pain and dysfunction are associated with this multifactorial disease characterized by the gradual degeneration of articular cartilage. The metabolic reprogramming of osteoarthritic chondrocytes to glycolysis instead of oxidative phosphorylation results in reduced ATP and lactate accumulation. When the glycolytic metabolite pyruvate is converted into lactate by lactate dehydrogenase A (LDHA), cartilage degeneration occurs. In the present study, we examined the chondroprotective effects of the LDHA inhibitor, oxamate on experimental OA rats. Methods: Anterior cruciate ligament transection (ACLT)-induced ACLT-rats received an intraarticular (IA) injection of oxamate once a week for 5 weeks from the 10 th to 14 th after surgery. Animals were divided into four groups as follows: Sham, ACLT, ACLT + oxamate (0.25mg/kg), and ACLT + oxamate (2.5mg/kg). The results showed that an IA injection of oxamate significantly reduced weight-bearing defects and knee swelling in ACLT-rats. Results: Histopathological analyses showed that oxamate caused significantly less cartilage degeneration than ACLT alone. Moreover, IA oxamate exerts hypertrophic effects in the chondrocyte of articular cartilage by inhibiting glucose-transporter 1, glucose-transporter 3, pyruvate kinase, LDHA, pyruvate dehydrogenase kinase 1, and pyruvate dehydrogenase kinase 2. Terminal deoxynucleotidyl transferase dUTP nick end labeling revealed that oxamate significantly reduced chondrocyte apoptosis in articular cartilage. Conclusions: We propose that oxamate is beneficial for patients with OA and is associated with regulating glycolysis-related protein expression. In future clinical applications, our findings will provide new insights of LHDA inhibitor, oxamate into delaying strategies for OA progression. osteoarthritis oxamate glycolysis chondrocytes lactate dehydrogenase A Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Several complex mechanisms induce osteoarthritis (OA), including the progressive erosion of articular cartilage, proteoglycan degradation, and disruption of the collagen network, which lead to the progressive destruction of joints and loss of function [ 1 ]. Previous studies showed that metabolic pathways require reprogramming to adapt to energy changes following inflammatory responses [ 2 ]. During the inflammation, cells preferentially use glycolysis to generate energy. After inflammation has resolved, oxidative phosphorylation (OXPHOS) is resumed to provide energy [ 3 ]. Chondrocytes are the only cell type in cartilage and are mainly responsible for regulating extracellular matrix (ECM) repair and synthesis [ 4 ]. As cartilage tissues are avascular, nutrients and oxygen must be diffused from synovial fluid and subchondral bone to the chondrocytes [ 5 ]. Chondrocyte metabolism occurs in a hypoxic environment, with glycolysis the major method of energy production [ 6 , 7 ]. Glucose transported to chondrocytes is converted to two pyruvate molecules and two ATP molecules in the glycolytic pathway. The glycolytic intermediate pyruvate can enter the tricarboxylic acid cycle (TCA cycle) via the pyruvate dehydrogenase (PDH) complex to generate 36 ATP molecules by mitochondrial OXPHOS. When changes in the cartilage tissue microenvironment occur, chondrocytes regulate metabolic pathways to adapt to environmental changes [ 8 ]. In normal chondrocytes, around 25% of ATP originates from mitochondrial OXPHOS. However, as OA is accompanied by mitochondria dysfunctions in the cartilage, OXPHOS capacity decreases, resulting in energy deficiency [ 9 ]. Additionally, OA causes changes in energy metabolism pathways in chondrocytes, which are characterized by enhanced glycolysis and decreased OXPHOS capacity [ 10 , 11 ]. Glycolysis-related proteins or enzymes, such as glucose transporters 1 (GLUT1) [ 12 ], pyruvate kinase M2 (PKM2) [ 13 ], and lactate dehydrogenase A (LDHA) [ 14 ], were reportedly upregulated in chondrocytes from patients with OA compared to healthy controls. The glycolytic intermediate, pyruvate, is converted into lactate by LDHA. Lactate, once considered an end product of glycolysis, has recently emerged as a critical regulator of OA development [ 15 , 16 ]. The inhibition of LDHA activity in chondrocytes by genetic manipulation (LDHA knockout) in mice, or pharmacological manipulation in vitro, has identified LDHA as a potential therapeutic target for OA treatment [ 14 ]. Oxamate is a pyruvate analogue that mainly acts as a competitive inhibitor of LDHA to block the conversion of pyruvate to lactate [ 17 ]. Studies in recent years have reported the correlations between glycolysis and various diseases, including neurodegenerative diseases [ 18 ], rheumatoid arthritis (RA) [ 19 ], cancer [ 20 ], and diabetes [ 21 ]. Oxamate can inhibit glycolysis while stimulating mitochondrial OXPHOS to promote differentiation in mouse osteoprogenitors and human bone marrow stromal cells (BMSCs) [ 22 ]. However, to our knowledge, no other studies have examined the effects of oxamate in the treatment of OA. The present study used LDHA inhibitors, oxamate to elucidate the role of glycolysis-related enzymes on ACLT-induced OA in rats. Mathods Animals and surgical technique for OA induction OA was induced in male Wistar rats (8 weeks old, body weight 260–280g) by anterior cruciate ligament transaction (ACLT) of the right knee; surgery was not performed on the left knee. This procedure was modified from the protocol described previously [ 23 , 24 ]. Each animal was administered a subcutaneous pre-operative injection of enrofloxacin (5 mg/kg) for prophylaxis. The animals were not immobilized after surgery and were allowed unrestricted cage activity. All rats were maintained in climate-controlled conditions on a 12-h light-dark cycle at 22–24°C at a relative humidity of 50–55%. Experimental design and oxamate treatment The rats were randomly allocated into the following four experimental groups: Group I: sham; rats received arthrotomy without ACLT and were administered 50 µl of physiologically normal saline by intra-articular administration. Group II: ACLT; animals that underwent ACLT and were administered 50 µl of physiologically normal saline by intra-articular administration. Group III: ACLT + 0.25 mg/kg oxamate; animals that underwent ACLT and were administered 0.25 mg/kg of oxamate (Thermo Fisher Scientific, Waltham, USA) by intra-articular administration once weekly for five consecutive weeks, beginning 10 weeks after surgery. Group IV: ACLT + 2.5 mg/kg oxamate: animals that underwent ACLT and were administered 0.25 mg/kg of oxamate by intra-articular administration once weekly for five consecutive weeks, beginning 10 weeks after surgery. Ultimately, all animals were sacrificed, and their knee joints were collected for histopathological and immunohistochemical analyses. In our preliminary observation, compared with the ACLT group, changes in the hind paw weight-bearing distribution had decreased significantly, but the knee joint swelling had decreased insignificantly in ACLT + oxamate 0.125 mg/kg ( n = 3). Therefore, we examine the effects of oxamate 0.25mg/kg and 2.5mg/kg on ACLT-rats. Test for Defects in Hind Limb Weight-bearing and Joint Width Measurement The effect of joint damage on the weight distribution in OA and contra-lateral knees was measured using an incapacitance meter tester (Singa Technology Corporation, Taipei, TW) that independently measured the weight bearing of each hind paw. Changes in the distribution of hind paw weight-bearing were used as an index of joint discomfort as described previously [ 25 , 26 ]. Briefly, changes in hind paw weight-bearing distribution between the contralateral control and OA limbs were used as an index of joint pain in the surgically induced OA knee. The rats were placed in an angled Plexiglass chamber positioned so that each hind paw rested on a separate force plate. The force exerted by each hind limb (measured in grams) was averaged over a 5-s period. Each data point was the mean of three, 5-s readings. The hind paw weight distribution was expressed as the differences in weight between the contralateral and ipsilateral limbs. The width of the knee joint was measured from the medial to the lateral aspects of the knee joint at approximately the level of the medial and lateral joint lines using a vernier caliper (AA847R, Aesculap, AG&CO, KG, Osnabrück, Germany). Changes in knee joint width, a measure of knee joint inflammation, were recorded weekly before and after ACLT for up to 26 weeks. Gross morphology and histopathological examination At week 26 after ACLT, the rats were sacrificed by deep anesthesia with sodium pentobarbital (50 mg/kg), then perfused intracardially with heparinized saline (200 ml/rat) followed by freshly prepared 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline, pH 7.4. The joints were sectioned 1 cm above and below the joint line, fixed in 10% neutral buffered formalin for 3 days, and then decalcified for 8 weeks in a buffered 4% ethylenediaminetetraacetic acid (EDTA) and formalin solution. The joints were then sectioned mid-sagittally, washed under running tap water, and paraffin-embedded using an automatic processor (Autotechnicon Mono 2; Technion Co., Chauncey, NY). Serial articular cartilage sections (1 µm) were cut on a rotatory microtome Microm HM340E (Walldorf, Germany) from the central weight-bearing surface of the femoral condyles and tibial plateau of both knees. Safranin-O/fast green staining was performed to assess the general morphology and matrix proteoglycan. Immediately after sacrifice, each knee was examined for gross morphologic changes in the cartilage lesions as described previously [ 27 ]. The articular cartilage was graded by microscopy according to the Osteoarthritis Research Society International (OARSI) system [ 28 ]. This system comprises six histological grades and four histological stages. The total score (score = grade × stage) ranges from 1 point (normal articular cartilage) to 24 points (no repair). Immunohistochemistry Cartilage specimens were processed for immunohistochemical analysis as described previously[ 26 , 29 ]. Briefly, 1 µm sections of paraffin-embedded specimens were placed on slides, deparaffinized with xylene, and dehydrated in an alcohol gradient. The antigen was retrieved by enzymatic digestion with proteinase K (20 mM) in Tris-EDTA buffer for 45 mins. The endogenous peroxidase activity then was quenched by 8-min incubation in 3% hydrogen peroxide. After washing three times for 8 mins in Tween-tris-buffered saline (TTBS), the sections were incubated in phosphate-buffered saline (PBS) containing 4% normal horse serum for 60 mines as a blocking agent for non-specific binding. The sections were incubated for 90 mines with biotinylated anti-rabbit or -mouse IgG (Vector Labs, Burlingame, CA) diluted 200-fold in 2% bovine serum albumin (BSA) in PBS. The sections were then treated using the avidin-biotin complex technique using an ABC kit (Vectastain A.B.C. kit; Vector Labs, Burlingame, CA). The images were viewed using a Leica DM6000 microscope (Leica, Heidelberg, Germany) and captured using a FLEXACAM C1 microscope camera (Leica, Heidelberg, Germany) The different antigens present in each cartilage specimens were quantified and estimated by determining the number of positively stained chondrocytes in the entire thickness of the cartilage, as described previously [ 30 ]. The cartilage was divided into six microscopic fields (three each in the superficial and deep zones) (magnification, 400×), and the results were averaged. For each OA specimen, before evaluation, the presence of an intact cartilage surface that could be detected and used as a marker for the morphometric analyses. The data was expressed as the percentage of chondrocytes showing positive staining for the antigen (cell score), with the maximum cartilage specimen scores of 100%. Each slide was reviewed by two independent readers blinded to the treatment groups. The data obtained from the medial and lateral femoral condyle and tibial plateau were considered together for the statistical analyses. Data and statistical analysis All continuous data are presented as means ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used to test differences among the means of various scores in the experimental groups and for scores with significant differences. To compare mean differences between treatment and sham groups, Student-Newman-Keuls post hoc tests were used. The trends in the changes in nociceptive behavior and knee joint width were tested using repeated-measures ANOVA. Differences with P < 0.05 were considered significant, including the four groups in the present study. Results Effects of intra-articular oxamate on ACLT-induced nociception In order to assess the effects of oxamate on OA-induced weight-bearing changes and knee swelling, oxamate in 0.25 mg/kg and 2.5 mg/kg concentrations was administered via intraarticular (IA) injection once a week from Week 10 to Week 14 after ACLT surgery. In addition to the pain and joint instability caused by uneven weight bearing, rats with osteoarthritis (OA) experience an inflammatory response that includes joint swelling. Like the animal in the ACLT-group the animals in the ACLT + oxamate group showed no obvious neurological defects after ia oxamate injection. As shown in Fig. 1 A, After ACLT surgery, weight-bearing changes increased in rats with time until before oxamate treatment in Week 10. The hind limbs of rats in the ACLT group (42.08 ± 2.40 g) were shown to have significantly greater weight-bearing changes than those in the sham group (1.41 ± 0.75 g). The comparison of the ACLT + oxamate 0.25 mg/kg group and the ACLT group found that differences after Week 16 were significant and persisted until Week 26. The comparison of the ACLT + oxamate 2.5 mg/kg group and the ACLT group found that differences after Week 16 were significant and persisted until Week 26. Based on the above results, oxamate at 0.25 mg/kg, and 2.5 mg/kg can effectively improve lower-limb weight-bearing distribution in the ACLT-induced OA model. As shown in Fig. 1 B, after ACLT surgery, knee joint swelling increased in rats with time until before oxamate treatment in Week 10. Rats in the ACLT group (0.65 ± 0.03 mm) exhibited significantly higher knee joint swelling than those in the sham group (0.02 ± 0.01 mm). The comparison of the ACLT + oxamate 0.25 mg/kg treatment group and the ACLT group found that differences after Week 16 were significant and persisted until Week 26. The comparison of the ACLT + oxamate 2.5mg/kg group and the ACLT group found that differences after Week 14 were significant and persisted until Week 26. Based on the above results, oxamate 0.125 mg/kg, 0.25mg/kg, and 2.5mg/kg can effectively improve knee joint swelling in the ACLT-induced OA model. Oxamate attenuates cartilage degradation in ACLT-rats Safranin O/Fast Green staining was used to assess the protective effects of oxamate in cartilage degradation/damage, and the Osteoarthritis Research Society International (OARSI) system was used to analyze knee joint tissues in the sham, ACLT, and ACLT + oxamate (0.25 mg/kg or 2.5mg/kg) groups. The results are shown in Fig. 2 A. Compared to the sham group, extracellular matrix (ECM) loss and severe damage to the articular surface were observed in the ACLT group. Although ECM loss was not alleviated in the ACLT + oxamate 0.25 mg/kg group, there was an improvement in articular surface damage. ECM loss and articular surface injury were improved in the ACLT + oxamate 2.5mg/kg group. Figure 2 B shows the quantitative analysis results by the OARSI system: The ACLT group (8.25 ± 1.73) scored significantly higher than the sham group (0.33 ± 0.33). Although the ACLT + oxamate 0.25 mg/kg group (4.80 ± 1.83, p = 0.205) had a lower OARSI score than the ACLT group, the difference was insignificant. The ACLT + oxamate 2.5 mg/kg group (2.20 ± 0.73) had a significantly lower OARSI score than the ACLT group. Based on the above results, treatment with oxamate at 2.5 mg/kg can significantly reduce cartilage ECM and articular surface injury in the ACLT-induced OA model. Oxamate affects glucose-transporter 1 (GLUT1) and GLUT3 expression in ACLT cartilage GLUT1 and GLUT3 are GLUT isoforms, and their expression indicates an increased glucose uptake, thereby promoting glycolysis. The results are shown in Fig. 3 A. The ACLT group exhibited increased GLUT1 protein expression in cartilage tissues (above the dotted line) compared to the sham group. Treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg can significantly decrease GLUT1 protein expression in ACLT-cartilage tissues. The quantitative results are shown in Fig. 3 B. GLUT1 protein expression was significantly higher in the ACLT group than in the sham group. After treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg, GLUT1 protein expression was significantly downregulated compared to that in the ACLT group. The above results showed that oxamate at 0.25 mg/kg and 2.5 mg/kg can decrease GLUT1 protein expression in ACLT-cartilage tissues. The results of GLUT3 protein expression are shown in Fig. 3 C. Compared to the sham group, GLUT3 protein expression was increased in cartilage tissues (above the dotted line) in the ACLT group. Treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg can significantly decrease GLUT3 protein expression in ACLT-cartilage tissues. The quantitative results are shown in Fig. 3 D. GLUT3 protein expression was significantly higher in the ACLT group than in the sham group. After treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg, GLUT3 protein expression was significantly downregulated compared to that in the ACLT group. The above results showed that oxamate at 0.25 mg/kg and 2.5 mg/kg can decrease GLUT3 protein expression in ACLT-cartilage tissues. Oxamate affects hexokinase II (HK-II) expression in ACLT cartilage HK-II is a major regulatory protein in glycolysis, and its expression can increase the conversion rate of glucose to glucose 6-phosphate (G6P), thereby promoting glycolysis. The results are shown in Fig. 4 A. Compared to the sham group, HK-II protein expression was increased in cartilage tissues (above the dotted line) in the ACLT group.Treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg can decrease HK-II protein expression in ACLT-cartilage tissues. The quantitative results are shown in Fig. 4 B. HK-II protein expression was significantly higher in the ACLT group than in the sham group. Although HK-II protein expression was downregulated after treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg compared to that in the ACLT group, the decreases were not significant. Oxamate affects pyruvate kinase M2 (PKM2) expression in ACLT cartilage PKM2 is a PK isoform that helps cells to obtain energy via glycolysis in a hypoxic environment. The results are shown in Fig. 4 C. Compared to the same group, PKM2 protein expression was increased in cartilage tissues (above the dotted line) in the ACLT group.Treatment with oxamate at 2.5 mg/kg can decrease PKM2 protein expression in ACLT-cartilage. The quantitative results are shown in Fig. 4 D. Compared to the sham group, PKM2 protein expression was significantly increased in the ACLT group. Although PKM2 protein expression was downregulated after treatment with oxamate at 0.25 mg/kg compared to that in the ACLT group (p = 0.318), the decrease was not significant. PKM2 protein expression was significantly downregulated after treatment with oxamate at 2.5 mg/kg compared to the ACLT group. Based on the above results, treatment with oxamate at 2.5 mg/kg can significantly decrease PKM2 protein expression in cartilage tissues in the ACLT-induced OA model. Oxamate affects lactate dehydrogenase A (LDHA) expression in ACLT cartilage LDHA can convert pyruvate synthesized during glycolysis to lactate in a hypoxic environment. The results are shown in Fig. 5 A. The ACLT group exhibited increased LDHA protein expression in cartilage tissues (above the dotted line) compared to the sham group. Treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg can decrease LDHA protein expression in ACLT-cartilage tissues. The quantitative results are shown in Fig. 5 B. LDHA protein expression was significantly higher in the ACLT group than in the sham group. After treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg, LDHA protein expression was significantly downregulated compared to that in the ACLT group. The above results showed that oxamate at 0.25 mg/kg and 2.5 mg/kg can decrease LDHA protein expression in ACLT-cartilage tissues. Oxamate affects pyruvate dehydrogenase kinase 1 (PDK1) and PDK2 expression in ACLT cartilage PDK1 and PDK2 are PDK isoforms with the primary function of inhibiting acetyl-CoA entry into the tricarboxylic acid cycle and decreasing OXPHOS. The results are shown in Fig. 6 A. Compared to the sham group, PDK1 protein expression was increased in cartilage tissues (above the dotted line) in the ACLT group. Treatment with oxamate at 2.5 mg/kg can decrease PDK1 protein expression in ACLT-cartilage tissues. The quantitative results are shown in Fig. 6 B. Compared to the sham group, PDK1 protein expression was significantly increased in the ACLT group. Although PDK1 protein expression was downregulated after treatment with oxamate at 0.25 mg/kg compared to that in the ACLT group (p = 0.171), the decrease was not significant. PDK1 protein expression was significantly downregulated after treatment with oxamate at 2.5 mg/kg compared to that in the ACLT group. Based on the above results, treatment with oxamate at 2.5 mg/kg can significantly decrease PDK1 protein expression in cartilage tissues in the ACLT-induced OA model. The results of PDK2 protein expression are shown in Fig. 6 A. Compared to the sham group, PDK2 protein expression was increased in cartilage tissues (above the dotted line) in the ACLT group. Treatment with oxamate at 0.25 mg/kg and 2.5mg/kg can decrease PDK2 protein expression in ACLT-cartilage tissues. The quantitative results are shown in Fig. 6 B. Compared to the sham group, PDK2 protein expression was significantly increased in the ACLT group. After treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg, PDK2 protein expression was significantly downregulated compared to that in the ACLT group. The above results showed that oxamate at 0.25 mg/kg and 2.5 mg/kg can decrease PDK2 protein expression in ACLT-cartilage tissues. Oxamate affects TUNEL expression in ACLT cartilage The results are shown in Fig. 7 A. The ACLT group exhibited increased TUNEL expression in cartilage tissues (above the dotted line) compared to the sham group. Treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg can significantly decrease TUNEL protein expression in ACLT-cartilage tissues. The quantitative results are shown in Fig. 7 B. TUNEL expression was significantly higher in the ACLT group than in the sham group. After treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg, TUNEL protein expression was significantly downregulated compared to that in the ACLT group. The above results showed that oxamate at 0.25 mg/kg and 2.5 mg/kg can decrease TUNEL protein expression in ACLT-cartilage tissues. Discussion Because articular cartilage lacks vascular, neural, and lymphatic tissues, nutrients and oxygen are transported by diffusion from synovial fluid secreted by synovial tissues. Oxamate is an LDHA inhibitor, and previous research has shown that it mainly regulates glycolysis when used for inhibiting cancer cell growth [ 31 , 32 ]. The present results indicated that IA administering oxamate to rats with ACLT-induced OA could significantly improve weight-bearing defects and knee joint swelling in their hind limbs. From histopathological observations, oxamate inhibits ACLT-induced cartilage degradation and chondrocyte apoptosis. At the same time, oxamate decreases the upregulation of glycolysis-related proteins such as GLUT1, GLUT3, HK-II, PKM2, LDHA, PDK1, and PDK2 in cartilage tissues in ACLT-induced OA. Based on clinical study, there is a 50% chance of developing knee OA 10–20 years after ACL injury [ 33 ]. Histopathological examination conducted in rat experiments revealed that OA induced by this surgery is caused by joint instability combined with intense pain, resulting in uneven weight bearing in the hind limbs and articular cartilage degeneration [ 34 ]. Several studies have demonstrated that the progression of OA is accompanied by nociceptive behaviors [ 35 – 37 ]. Previous in vivo studies also have indicated that the ACLT-induced OA model allows clear observation of mechanical allodynia and changes in Weight-bearing distribution[ 29 , 38 , 39 ]. Moreover, there will be swelling accompanying the injured knee joint with disease progression after ACLT [ 29 , 39 ]. The present study found that an IA injection of oxamate after surgery could attenuate pain behavior due to weight-bearing defects in the hind limb (Fig. 1 A). In addition to pain and joint instability, patients with OA exhibit inflammatory responses that include joint swelling and synovitis [ 40 ]. Previous studies have suggested that ACLT surgery induces early OA in rats, and its joint pathology increases joint diameter, cartilage degeneration, synovitis, and chondrocyte death [ 41 – 43 ]. The results of this study were similar to previous studies: There was significant knee joint swelling (Fig. 1 B) and cartilage degeneration (Fig. 2 ) in the ACLT group, and an IA injection with oxamate could significantly improve the aforementioned pathological phenomenon. Up to the present, GLUTs have 14 isoforms [ 44 ]. Their function is to transport extracellular glucose into cells, thereby providing a source of glucose for glycolysis. Studies have found that GLUT1, GLUT3, and GLUT9 are expressed in normal chondrocytes, with GLUT1 and GLUT3 being extremely sensitive to hypoxic environments [ 45 , 46 ]. In 2013, Vázquez-Mosquera et al. proved that GLUT1 and GLUT3 expressions in cartilage tissues in patients with OA were higher than in healthy people, but these differences were insignificant [ 46 , 47 ]. This study showed that GLUT1 and GLUT3 expressions in cartilage tissue were significantly upregulated in the ACLT-induced OA group compared to those in the naïve group (Fig. 3 ). Previous research found that while increasing glucose transport helps cells obtain energy, it also causes oxidative stress and inflammation [ 48 ]. Upregulation of oxidative stress and inflammation can damage chondrocytes and ultimately exacerbate OA [ 49 ]. According to our findings, an IA injection of oxamate inhibits ACLT-induced GLUT1 and GLUT3 expressions in chondrocytes (Figs. 3 ). We hypothesize that oxamate downregulates the expression of these 2 GLUTs, which may regulate more efficient energy generation methods (discussed after this section) and reduce oxidative stress and inflammation, both of which have protective effects in OA. In an anaerobic phase, chondrocytes in OA synthesize pyruvate via glycolysis and LDHA converts pyruvate to lactate. This conversion pathway can generate two ATP molecules to provide energy, but lactate accumulation causes an acidic microenvironment [ 50 ]. Studies have shown that low extracellular pH can regulate the expression of ECM-degrading enzymes. Moreover, extracellular pH < 7.1 inhibits ECM synthesis while increasing the expression of ECM-degrading enzymes, resulting in cartilage degeneration [ 51 – 53 ]. In addition, the acidic environment in the OA articular cavity causes pain [ 54 ]. In 2020, Arra et al. used an ex vivo OA model to demonstrate that inflammation causes chondrocyte glycolysis, elevates LDHA, and decreases OXPHOS [ 55 ]. Downregulation of LDHA can reduce the expression of the ECM-degrading enzyme MMP13 and protects cartilage tissues in OA animal experiments [ 55 ]. Our findings also showed that in ACLT-induced OA rats, LDHA protein expression was upregulated in chondrocytes (Fig. 5 ), which was accompanied by ECM loss and surface damage in cartilage tissues (Fig. 2 ). Following IA administration with the LHDA inhibitor (oxamate), ECM loss and cartilage surface injury were significantly improved. Simultaneously, IHC staining results revealed that an IA injection of oxamate could significantly inhibit LDHA protein expression in ACLT-induced OA (Fig. 5 ). Previous reports and the present results found that LHDA inhibition and downregulated protein expression protect joints in OA. Normal chondrocytes receive 75% of their energy from glycolysis, but mitochondria receive 25% of their energy from OXPHOS [ 9 ]. An oxygen gradient microenvironment exists in cartilage tissues, and chondrocytes in the deepest part can obtain 1% oxygen. Hence, both glycolysis and OXPHOS occur in chondrocytes [ 56 ]. A study revealed that most chondrocytes’ energy demands are met by glycolysis rather than OXPHOS [ 56 ]. Although OXPHOS provides 25% of ATP to chondrocytes, this pathway can synthesize 36 ATP molecules from a single glucose molecule and its energy generation efficiency is far greater than glycolysis, which can only synthesize 2 ATP molecules from 1 glucose molecule [ 57 ]. In 2015, Qu et al. demonstrated that while glycolysis was increased in OA chondrocytes, ATP energy acquisition was decreased, inhibiting chondrocyte proliferation, differentiation, and cell viability [ 58 ]. We used oxamate to inhibit OA-induced LHDA protein upregulation. This might allow more intermediate product of glycolysis, pyruvate to enter mitochondrial OXPHOS and produce more energy for repairing damaged chondrocytes and decreasing apoptosis. Therefore, oxamate can improve cartilage degeneration and chondrocyte death. The pyruvate dehydrogenase complex (PDC) is essential in aerobic metabolism because it catalyzes pyruvate entry into mitochondria for OXPHOS [ 59 ]. PDC activity is negatively regulated by PDK. In pathological conditions (e.g., diabetes, cancer, and sepsis), PDK activation inhibits PDC activity and prevents cytoplasmic pyruvate from entering mitochondria for OXPHOS, resulting in decreased ATP synthesis [ 60 ]. The previous study has found that PDK decreases PDC activity, it increases the glucose metabolites pyruvate and lactate while decreasing mitochondrial OXPHOS [ 61 ]. Our experiments found that ACLT upregulated PDK1 and PDK2 in chondrocytes (Fig. 6 ), which may inhibit PDC activity, thereby affecting pyruvate entry into the mitochondrial OXPHOS for ATP synthesis. The previous study also indicate that a hypoxic environment can increases hypoxia-inducible factor 1-alpha (HIF-1α) expression and activates PDK1 [ 62 ]. This results in the reprogramming of glucose metabolism from OXPHOS to aerobic glycolysis and a decrease in ATP synthesis [ 63 ]. Yudoh et al. (2004) demonstrated that HIF-1α expression is elevated in the cartilage tissues of patients with OA and contributes to cartilage degeneration [ 64 ]. Therefore, we believe that ACLT-induced PDK upregulation is caused by HIF-1α. Its primary function of HK is to convert phosphorylated glucose to glucose 6-phosphate [ 65 ]. HK-IIcan reprogram tumor cells’ metabolisms to aerobic glycolysis [ 66 , 67 ]. A study revealed that HK-II expression in peripheral blood mononuclear cells is higher in patients with OA than in healthy people [ 68 ]. This study found that ACLT upregulates HK-II expression in chondrocytes (Fig. 4 A and 4 B). Many studies have proved that transforming growth factor beta (TGF-β) plays an important role in OA progression [ 69 – 71 ]. A previous study found that TGF-β1 upregulates HK-II protein expression in OA chondrocytes and induces aerobic glycolysis, increases glucose consumption and lactate synthesis, and decreases ATP synthesis simultaneously [ 72 ]. Our previous studies also found that ACLT increases TGF-β1 in chondrocytes [ 26 , 29 ]. Therefore, we suggest that ACLT-induced HK-II upregulation may be regulated by TGF-β. Pyruvate kinase(PK) has two isoforms (PKM1 and PKM2) and mainly mediates the final step in glycolysis, which converts phosphoenolpyruvate to pyruvate, and this step generates one ATP [ 73 ]. Previous studies proved that PKM2 expression in the cartilage of patients with OA was higher than that of healthy people and that PKM2 knockdown could downregulate GLUT1, HIF-1α, and LDHA expressions, inhibit OA chondrocyte proliferation, and promote apoptosis [ 13 ]. The present study found that the PKM2 protein is significantly increased in chondrocytes when OA occurs and that oxamate injection significantly downregulates the expression of this protein (Fig. 4 C and 4 D). This could be because oxamate inhibits LDHA, PDK1 and PDK2 expressions in the ACLT group while partially restoring ATP synthesis in the OXPHOS pathway. Previous studies have proved that oxamate can promote bone formation and bone strength in mice [ 22 ], but its protective mechanisms in OA are unknown. This study found that an IA injection of oxamate inhibits ACLT-induced LDHA, PDK1, and PDK2 upregulation and may promote OXPHOS restoration to increase ATP synthesis, assist in chondrocyte repair, and decrease apoptosis. However, as long as ATP supply is available in sufficient quantities, glycolysis-related proteins such as GLUT1, GLUT3, HK-II, and PKM2 may be indirectly regulated by oxamate in ACLT-induced OA. Conclusions The results of this study support the fact that oxamate can regulate the expression of glucose metabolism proteins and partially restore OXPHOS to provide more ATP to protect chondrocytes. We believe that an IA injection of oxamate has protective effects in OA and has potential for development. Abbreviations ACLT Anterior cruciate ligament transection ECM Extracellular matrix GLUT1 Glucose-transporter 1 GLUT3 Glucose-transporter 3 HK-II Hexokinase II IA Intraarticular LDHA Lactate dehydrogenase A OA Osteoarthritis OXPHOS Oxidative phosphorylation PDC Pyruvate dehydrogenase complex PDH Pyruvate dehydrogenase PDK1 Pyruvate dehydrogenase kinase 1 PDK2 Pyruvate dehydrogenase kinase 2 PK Pyruvate kinase PKM2 Pyruvate kinase M2 TCA cycle Tricarboxylic acid cycle TUNEL Terminal deoxynucleotidyl transferase dutp nick-end labeling Declarations Ethics approval and consent to participate The animal experiments were performed according to the Guiding Principles in the Care and Use of Animals, as approved by the Council of the American Physiology Society, and were approved by the National Sun Yat-sen University Animal Care and Use Committee (approval no.11028). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Availability of data and materials The datasets supporting the conclusions of this study are included within the article. Funding The study was supported by the National Science and Technology Council, Taiwan (NSCT 110-2314-B-843-001; NSCT 111-2314-B-843-001; NSCT 109-2314-B-075) and partly supported by Ping-Tung Christian Hospital. Authors’ contribution Zhi-Hong Wen: Conceptualization, Writing-Original draft preparation and Formal analysis; Chun-Sung Sung: Conceptualization, Writing-Reviewing and Editing, Formal analysis and Funding acquisition; Yen-Hsuan Jean: Conceptualization, Writing-Original draft preparation, Formal analysis and Funding acquisition; Sung-Chun Lin: Writing-Reviewing and Editing; Zhi-Kang Yao: Software and Validation; Yu-Wei Liu: Methodology and Investigation; Yu-Yan Wu: Methodology and Investigation; Yu-Cheng Lai: Software, Validation and Formal analysis; Wu-Fu Chen: Writing- Reviewing and Editing. Hsin-Tzu Liu: Visualization. Acknowledgments The authors also thank editage (www.editage.com.tw) for the English language review. References Walsh MC, Kim N, Kadono Y, Rho J, Lee SY, Lorenzo J, Choi Y: OSTEOIMMUNOLOGY: Interplay Between the Immune System and Bone Metabolism . Annu Rev Immunol 2006, 24 (1):33–63. Hu C, Xuan Y, Zhang X, Liu Y, Yang S, Yang K: Immune cell metabolism and metabolic reprogramming . Mol Biol Rep 2022, 49 (10):9783–9795. Soto-Heredero G, Gomez de Las Heras MM, Gabande-Rodriguez E, Oller J, Mittelbrunn M: Glycolysis - a key player in the inflammatory response . FEBS J 2020, 287 (16):3350–3369. Akkiraju H, Nohe A: Role of Chondrocytes in Cartilage Formation, Progression of Osteoarthritis and Cartilage Regeneration . J Dev Biol 2015, 3 (4):177–192. Le Clanche S, Bonnefont-Rousselot D, Sari-Ali E, Rannou F, Borderie D: Inter-relations between osteoarthritis and metabolic syndrome: A common link? Biochimie 2016, 121 :238–252. Lane RS, Fu Y, Matsuzaki S, Kinter M, Humphries KM, Griffin TM: Mitochondrial respiration and redox coupling in articular chondrocytes . Arthritis Res Ther 2015, 17 :54. Nishida T, Kubota S, Aoyama E, Takigawa M: Impaired glycolytic metabolism causes chondrocyte hypertrophy-like changes via promotion of phospho-Smad1/5/8 translocation into nucleus . Osteoarthritis Cartilage 2013, 21 (5):700–709. Zheng L, Zhang Z, Sheng P, Mobasheri A: The role of metabolism in chondrocyte dysfunction and the progression of osteoarthritis . Ageing Research Reviews 2021, 66 :101249. Wu X, Fan X, Crawford R, Xiao Y, Prasadam I: The Metabolic Landscape in Osteoarthritis . Aging Dis 2022, 13 (4):1166–1182. Mobasheri A, Rayman MP, Gualillo O, Sellam J, van der Kraan P, Fearon U: The role of metabolism in the pathogenesis of osteoarthritis . Nature Reviews Rheumatology 2017, 13 (5):302–311. Kudelko M, Chan CW, Sharma R, Yao Q, Lau E, Chu IK, Cheah KS, Tanner JA, Chan D: Label-Free Quantitative Proteomics Reveals Survival Mechanisms Developed by Hypertrophic Chondrocytes under ER Stress . Journal of proteome research 2016, 15 (1):86–99. Pfander D, Cramer T, Swoboda B: Hypoxia and HIF-1alpha in osteoarthritis . Int Orthop 2005, 29 (1):6–9. Yang X, Chen W, Zhao X, Chen L, Li W, Ran J, Wu L: Pyruvate Kinase M2 Modulates the Glycolysis of Chondrocyte and Extracellular Matrix in Osteoarthritis . DNA Cell Biol 2018, 37 (3):271–277. Arra M, Swarnkar G, Ke K, Otero JE, Ying J, Duan X, Maruyama T, Rai MF, O'Keefe RJ, Mbalaviele G et al : LDHA-mediated ROS generation in chondrocytes is a potential therapeutic target for osteoarthritis . Nat Commun 2020, 11 (1):3427. Maneiro E, Martin MA, de Andres MC, Lopez-Armada MJ, Fernandez-Sueiro JL, del Hoyo P, Galdo F, Arenas J, Blanco FJ: Mitochondrial respiratory activity is altered in osteoarthritic human articular chondrocytes . Arthritis Rheum 2003, 48 (3):700–708. Richardson SM, Hoyland JA, Mobasheri R, Csaki C, Shakibaei M, Mobasheri A: Mesenchymal stem cells in regenerative medicine: opportunities and challenges for articular cartilage and intervertebral disc tissue engineering . J Cell Physiol 2010, 222 (1):23–32. Zhai X, Yang Y, Wan J, Zhu R, Wu Y: Inhibition of LDH-A by oxamate induces G2/M arrest, apoptosis and increases radiosensitivity in nasopharyngeal carcinoma cells . Oncol Rep 2013, 30 (6):2983–2991. Tang BLJJoCP: Glucose , glycolysis , and neurodegenerative diseases . 2020, 235 (11):7653–7662. Abboud G, Choi S-C, Kanda N, Zeumer-Spataro L, Roopenian DC, Morel LJFii: Inhibition of glycolysis reduces disease severity in an autoimmune model of rheumatoid arthritis . 2018, 9 :1973. Al-Ziaydi AG, Al-Shammari AM, Hamzah MI, Kadhim HS, Jabir MSJV: Newcastle disease virus suppress glycolysis pathway and induce breast cancer cells death . 2020, 31 (3):341–348. Guo X, Li H, Xu H, Woo S, Dong H, Lu F, Lange AJ, Wu C: Glycolysis in the control of blood glucose homeostasis . Acta Pharmaceutica Sinica B 2012, 2 (4):358–367. Hollenberg AM, Smith CO, Shum LC, Awad H, Eliseev RA: Lactate Dehydrogenase Inhibition With Oxamate Exerts Bone Anabolic Effect . J Bone Miner Res 2020, 35 (12):2432–2443. Stoop R, Buma P, van der Kraan PM, Hollander AP, Billinghurst RC, Meijers TH, Poole AR, van den Berg WB: Type II collagen degradation in articular cartilage fibrillation after anterior cruciate ligament transection in rats . Osteoarthritis Cartilage 2001, 9 (4):308–315. Jean YH, Wen ZH, Chang YC, Hsieh SP, Tang CC, Wang YH, Wong CS: Intra-articular injection of the cyclooxygenase-2 inhibitor parecoxib attenuates osteoarthritis progression in anterior cruciate ligament-transected knee in rats: role of excitatory amino acids . Osteoarthritis Cartilage 2007, 15 (6):638–645. Bove SE, Laemont KD, Brooker RM, Osborn MN, Sanchez BM, Guzman RE, Hook KE, Juneau PL, Connor JR, Kilgore KS: Surgically induced osteoarthritis in the rat results in the development of both osteoarthritis-like joint pain and secondary hyperalgesia . Osteoarthritis and Cartilage 2006, 14 (10):1041–1048. Wen ZH, Lin YY, Chang YC, Tang CC, Hsieh SP, Lee HP, Sung CS, Chen WF, Lee CH, Hsuan Jean Y: The COX-2 inhibitor etoricoxib reduces experimental osteoarthritis and nociception in rats: The roles of TGF-beta1 and NGF expressions in chondrocytes . Eur J Pain 2020, 24 (1):209–222. Fernandes JC, Martel-Pelletier J, Otterness IG, Lopez-Anaya A, Mineau F, Tardif G, Pelletier JP: Effects of tenidap on canine experimental osteoarthritis. I. Morphologic and metalloprotease analysis . Arthritis Rheum 1995, 38 (9):1290–1303. Pritzker KP, Gay S, Jimenez SA, Ostergaard K, Pelletier JP, Revell PA, Salter D, van den Berg WB: Osteoarthritis cartilage histopathology: grading and staging . Osteoarthritis Cartilage 2006, 14 (1):13–29. Wen ZH, Tang CC, Chang YC, Huang SY, Lin YY, Hsieh SP, Lee HP, Lin SC, Chen WF, Jean YH: Calcitonin attenuates cartilage degeneration and nociception in an experimental rat model of osteoarthritis: role of TGF-beta in chondrocytes . Sci Rep 2016, 6 :28862. Boileau C, Martel-Pelletier J, Brunet J, Schrier D, Flory C, Boily M, Pelletier JP: PD-0200347, an alpha2delta ligand of the voltage gated calcium channel, inhibits in vivo activation of the Erk1/2 pathway in osteoarthritic chondrocytes: a PKCalpha dependent effect . Ann Rheum Dis 2006, 65 (5):573–580. Fiume L, Vettraino M, Manerba M, Di Stefano G: Inhibition of lactic dehydrogenase as a way to increase the anti-proliferative effect of multi-targeted kinase inhibitors . Pharmacol Res 2011, 63 (4):328–334. Zhao Z, Han F, Yang S, Wu J, Zhan W: Oxamate-mediated inhibition of lactate dehydrogenase induces protective autophagy in gastric cancer cells: involvement of the Akt-mTOR signaling pathway . Cancer Lett 2015, 358 (1):17–26. Louboutin H, Debarge R, Richou J, Selmi TA, Donell ST, Neyret P, Dubrana F: Osteoarthritis in patients with anterior cruciate ligament rupture: a review of risk factors . Knee 2009, 16 (4):239–244. Tawonsawatruk T, Sriwatananukulkit O, Himakhun W, Hemstapat W: Comparison of pain behaviour and osteoarthritis progression between anterior cruciate ligament transection and osteochondral injury in rat models . Bone Joint Res 2018, 7 (3):244–251. Litwic A, Edwards MH, Dennison EM, Cooper C: Epidemiology and burden of osteoarthritis . Br Med Bull 2013, 105 :185–199. Neogi T: The epidemiology and impact of pain in osteoarthritis . Osteoarthritis Cartilage 2013, 21 (9):1145–1153. Bartley EJ, Palit S, Staud R: Predictors of Osteoarthritis Pain: the Importance of Resilience . Curr Rheumatol Rep 2017, 19 (9):57. Wen ZH, Tang CC, Chang YC, Huang SY, Chen CH, Wu SC, Hsieh SP, Hsieh CS, Wang KY, Lin SY et al : Intra-articular injection of the selective cyclooxygenase-2 inhibitor meloxicam (Mobic) reduces experimental osteoarthritis and nociception in rats . Osteoarthritis Cartilage 2013, 21 (12):1976–1986. Kao JH, Lin SH, Lai CF, Lin YC, Kong ZL, Wong CS: Shea Nut Oil Triterpene Concentrate Attenuates Knee Osteoarthritis Development in Rats: Evidence from Knee Joint Histology . PLoS One 2016, 11 (9):e0162022. Bijlsma JW, Berenbaum F, Lafeber FP: Osteoarthritis: an update with relevance for clinical practice . Lancet 2011, 377 (9783):2115–2126. Barbosa GM, Cunha JE, Cunha TM, Martinho LB, Castro P, Oliveira FFB, Cunha FQ, Ramalho FS, Salvini TF: Clinical-like cryotherapy improves footprint patterns and reduces synovial inflammation in a rat model of post-traumatic knee osteoarthritis . Sci Rep 2019, 9 (1):14518. Yang Y, Li P, Zhu S, Bi R: Comparison of early-stage changes of osteoarthritis in cartilage and subchondral bone between two different rat models . PeerJ 2020, 8 :e8934. Pickarski M, Hayami T, Zhuo Y, Duong LT: Molecular changes in articular cartilage and subchondral bone in the rat anterior cruciate ligament transection and meniscectomized models of osteoarthritis . BMC Musculoskelet Disord 2011, 12 :197. Navale AM, Paranjape AN: Glucose transporters: physiological and pathological roles . Biophys Rev 2016, 8 (1):5–9. Mobasheri A, Neama G, Bell S, Richardson S, Carter SD: Human articular chondrocytes express three facilitative glucose transporter isoforms: GLUT1, GLUT3 and GLUT9 . Cell Biol Int 2002, 26 (3):297–300. Vannucci SJ, Seaman LB, Vannucci RC: Effects of hypoxia-ischemia on GLUT1 and GLUT3 glucose transporters in immature rat brain . J Cereb Blood Flow Metab 1996, 16 (1):77–81. Vázquez-Mosquera M, Rego-Pérez I, Soto-Hermida A, Fernández-Moreno M, Fernández-Tajes J, Cortés-Pereira E, Relaño-Fernández S, Oreiro-Villar N, Fernández-López C, Blanco FJO et al : New insights into the molecular basis of the metabolic alterations in the osteoarthritis (OA) disease . 2013, 21 :S170. Rosa SC, Goncalves J, Judas F, Mobasheri A, Lopes C, Mendes AF: Impaired glucose transporter-1 degradation and increased glucose transport and oxidative stress in response to high glucose in chondrocytes from osteoarthritic versus normal human cartilage . Arthritis Res Ther 2009, 11 (3):R80. Goldring MB: Update on the biology of the chondrocyte and new approaches to treating cartilage diseases . Best Pract Res Clin Rheumatol 2006, 20 (5):1003–1025. Zheng L, Zhang Z, Sheng P, Mobasheri A: The role of metabolism in chondrocyte dysfunction and the progression of osteoarthritis . Ageing Res Rev 2021, 66 :101249. Konttinen YT, Mandelin J, Li TF, Salo J, Lassus J, Liljestrom M, Hukkanen M, Takagi M, Virtanen I, Santavirta S: Acidic cysteine endoproteinase cathepsin K in the degeneration of the superficial articular hyaline cartilage in osteoarthritis . Arthritis Rheum 2002, 46 (4):953–960. Razaq S, Wilkins RJ, Urban JP: The effect of extracellular pH on matrix turnover by cells of the bovine nucleus pulposus . Eur Spine J 2003, 12 (4):341–349. Wilkins RJ, Hall AC: Control of matrix synthesis in isolated bovine chondrocytes by extracellular and intracellular pH . J Cell Physiol 1995, 164 (3):474–481. Hunter DJ, McDougall JJ, Keefe FJ: The symptoms of osteoarthritis and the genesis of pain . Rheum Dis Clin North Am 2008, 34 (3):623–643. Arra M, Swarnkar G, Ke K, Otero JE, Ying J, Duan X, Maruyama T, Rai MF, O’Keefe RJ, Mbalaviele G et al : LDHA-mediated ROS generation in chondrocytes is a potential therapeutic target for osteoarthritis . Nature Communications 2020, 11 (1):3427. Kan S, Duan M, Liu Y, Wang C, Xie J: Role of Mitochondria in Physiology of Chondrocytes and Diseases of Osteoarthritis and Rheumatoid Arthritis . Cartilage 2021, 13 (2_suppl):1102S-1121S. Bonora M, Patergnani S, Rimessi A, De Marchi E, Suski JM, Bononi A, Giorgi C, Marchi S, Missiroli S, Poletti F et al : ATP synthesis and storage . Purinergic Signal 2012, 8 (3):343–357. Qu J, Lu D, Guo H, Miao W, Wu G, Zhou M: PFKFB3 modulates glycolytic metabolism and alleviates endoplasmic reticulum stress in human osteoarthritis cartilage . Clin Exp Pharmacol Physiol 2016, 43 (3):312–318. Smolle M, Prior AE, Brown AE, Cooper A, Byron O, Lindsay JG: A new level of architectural complexity in the human pyruvate dehydrogenase complex . J Biol Chem 2006, 281 (28):19772–19780. Wang X, Shen X, Yan Y, Li H: Pyruvate dehydrogenase kinases (PDKs): an overview toward clinical applications . Biosci Rep 2021, 41 (4). McFate T, Mohyeldin A, Lu H, Thakar J, Henriques J, Halim ND, Wu H, Schell MJ, Tsang TM, Teahan O et al : Pyruvate dehydrogenase complex activity controls metabolic and malignant phenotype in cancer cells . J Biol Chem 2008, 283 (33):22700–22708. Kim JW, Tchernyshyov I, Semenza GL, Dang CV: HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia . Cell Metab 2006, 3 (3):177–185. Jeoung NH: Pyruvate Dehydrogenase Kinases: Therapeutic Targets for Diabetes and Cancers . Diabetes Metab J 2015, 39 (3):188–197. Yudoh K, Nakamura H, Masuko-Hongo K, Kato T, Nishioka K: Catabolic stress induces expression of hypoxia-inducible factor (HIF)-1 alpha in articular chondrocytes: involvement of HIF-1 alpha in the pathogenesis of osteoarthritis . Arthritis Res Ther 2005, 7 (4):R904-914. Roberts DJ, Miyamoto S: Hexokinase II integrates energy metabolism and cellular protection: Akting on mitochondria and TORCing to autophagy . Cell Death Differ 2015, 22 (2):248–257. Bustamante MF, Oliveira PG, Garcia-Carbonell R, Croft AP, Smith JM, Serrano RL, Sanchez-Lopez E, Liu X, Kisseleva T, Hay N et al : Hexokinase 2 as a novel selective metabolic target for rheumatoid arthritis . Ann Rheum Dis 2018, 77 (11):1636–1643. Ciscato F, Ferrone L, Masgras I, Laquatra C, Rasola A: Hexokinase 2 in Cancer: A Prima Donna Playing Multiple Characters . Int J Mol Sci 2021, 22 (9). Zhou KL, Zhu ZH, Zhou JP, Zhao JJ, Zhang Y, Jiang B: Increased hexokinase-2 as a novel biomarker for the diagnosis and correlating with disease severity in rheumatoid arthritis . Medicine (Baltimore) 2021, 100 (25):e26504. Blaney Davidson EN, van der Kraan PM, van den Berg WB: TGF-beta and osteoarthritis . Osteoarthritis Cartilage 2007, 15 (6):597–604. Shen J, Li S, Chen D: TGF-beta signaling and the development of osteoarthritis . Bone Res 2014, 2. van der Kraan PM: Differential Role of Transforming Growth Factor-beta in an Osteoarthritic or a Healthy Joint . J Bone Metab 2018, 25 (2):65–72. Wang C, Silverman RM, Shen J, O'Keefe RJ: Distinct metabolic programs induced by TGF-beta1 and BMP2 in human articular chondrocytes with osteoarthritis . J Orthop Translat 2018, 12 :66–73. Zahra K, Dey T, Ashish, Mishra SP, Pandey U: Pyruvate Kinase M2 and Cancer: The Role of PKM2 in Promoting Tumorigenesis . Front Oncol 2020, 10 :159. Tables Table 1 Primary antibodies used in the immunohistochemical analyses in the present study Primary Antibody Host Supplier Catalog # Dilution Ratio GLUT1 Rabbit abcam ab652 1:1000 GLUT3 Rabbit Biorbyt orb10727 1:1000 HK II Rabbit GeneTex gtx111525 1:3000 PKM2 Rabbit Cell Signaling Tecnology 4053s 1:800 PDK1 Mouse abcam ab110025 1:1000 PDK2 Rabbit abcam ab68164 1:800 LDHA Rabbit Novus NBP2-67483 1:500 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. 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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-2540780","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":172869407,"identity":"118f7bac-106b-4210-b837-14bd70d233d7","order_by":0,"name":"Zhi-Hong Wen","email":"","orcid":"","institution":"National Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhi-Hong","middleName":"","lastName":"Wen","suffix":""},{"id":172869410,"identity":"7c1e1d9b-7dfc-4fed-9c2c-3ae11663cee2","order_by":1,"name":"Chun-Sung Sung","email":"","orcid":"","institution":"Taipei Veterans General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chun-Sung","middleName":"","lastName":"Sung","suffix":""},{"id":172869414,"identity":"033db87b-2594-463c-aaec-6a1bcb4fb363","order_by":2,"name":"Sung-Chun Lin","email":"","orcid":"","institution":"Pingtung Christian Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sung-Chun","middleName":"","lastName":"Lin","suffix":""},{"id":172869416,"identity":"173eb746-c27c-4bb3-a8b0-b675b4b91d36","order_by":3,"name":"Zhi-Kang Yao","email":"","orcid":"","institution":"Kaohsiung Veterans General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhi-Kang","middleName":"","lastName":"Yao","suffix":""},{"id":172869417,"identity":"23cf8f10-0b0c-40c8-8f42-c1b992a38b01","order_by":4,"name":"Yu-Cheng Lai","email":"","orcid":"","institution":"National Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu-Cheng","middleName":"","lastName":"Lai","suffix":""},{"id":172869419,"identity":"5507fb55-1352-46a8-94b4-f9bb9fabcc75","order_by":5,"name":"Yu-Wei Liu","email":"","orcid":"","institution":"National Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu-Wei","middleName":"","lastName":"Liu","suffix":""},{"id":172869421,"identity":"9eed649a-4ed8-435e-99ac-9dd5b2ebdcb1","order_by":6,"name":"Yu-Yan Wu","email":"","orcid":"","institution":"National Sun Yat-sen University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu-Yan","middleName":"","lastName":"Wu","suffix":""},{"id":172869422,"identity":"4f859e0d-c262-452a-a826-631d50013a0b","order_by":7,"name":"Hsin-Tzu Liu","email":"","orcid":"","institution":"Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hsin-Tzu","middleName":"","lastName":"Liu","suffix":""},{"id":172869423,"identity":"08a5488b-e7e5-4d49-b4a1-825834564bc1","order_by":8,"name":"Wu-Fu Chen","email":"","orcid":"","institution":"Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wu-Fu","middleName":"","lastName":"Chen","suffix":""},{"id":172869424,"identity":"74881f94-f262-4337-83ad-4d168d78e01d","order_by":9,"name":"Yen-Hsuan Jean","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYBAC9gZU1gEegloYwQoTgJjnAEwLM4hhQIQWiQSwFgbCWtqbnz1g/GGXJx/5OvnDD4Y7Mubs/QeYCyr+4NbSc8zcgCEhudjwdu42yR6GZzyWPYcZmGecwWPLjAQzCYYE5sSNs3O3MfAwHOYxuJHMwMzbhkfL/OffgFrqEzfOPLv54x+QlvuPgVr+4dYiOIMHZMvhxPkSvBukIbYwA7U04NYizZNTJpGQdjxxA0/uNmkZA6CWM8lA8pgxTi187Me3SXywqU6c3w502JuKw/YGxw8+fMxTI4dTCxgkALHBARAL6pwD+NVDgXwDUcpGwSgYBaNgJAIA6IJSMLY9aJwAAAAASUVORK5CYII=","orcid":"","institution":"Pingtung Christian Hospital","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yen-Hsuan","middleName":"","lastName":"Jean","suffix":""}],"badges":[],"createdAt":"2023-02-02 02:59:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2540780/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2540780/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":32533340,"identity":"3b0dcfec-ba88-47f3-964e-c84c9a992bc7","added_by":"auto","created_at":"2023-02-06 14:46:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":40015,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of intraarticular oxamate injection on ACLT-induced OA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of oxamate on (A) ACLT-induced hind limb weight-bearing deficits and (B) knee swelling were studied over time. Rats in the ACLT + oxamate groups were intraarticularly injected with oxamate (0.25 mg/kg, or 2.5 mg/kg per week) from the 10\u003csup\u003eth\u003c/sup\u003e to the 14\u003csup\u003eth\u003c/sup\u003e week after ACLT. Data are expressed as means ± SEM for each group. (* p \u0026lt; 0.05, compared with the sham group; # p \u0026lt; 0.05, compared with the ACLT group).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2540780/v1/74fbb9df9e45cead3a659f9b.png"},{"id":32535532,"identity":"567cdd30-3df4-4ca4-b2df-27c77c4e1072","added_by":"auto","created_at":"2023-02-06 15:02:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":453897,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological evaluation of knee joints after oxamate treatment in ACLT-rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Histological sections of knee joints from the sham, ACLT, and ACLT + oxamate (0.25 mg/kg or 2.5 mg/kg) groups were stained with Safranin O/Fast Green. (B) Histopathological quantitive changes in the knee joints of the four studied groups were evaluated using the OARSI scoring system. The histogram shows the OARSI scores of the sham, ACLT, and ACLT + oxamate (0.25 mg/kg or 2.5 mg/kg) groups. Data are expressed as means ± SEM for each group. The scale bar represents 100 μm. OARSI, Osteoarthritis Research Society International. (* p \u0026lt; 0.05, compared with the sham group; # p \u0026lt; 0.05, compared with the ACLT group).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2540780/v1/1930d6024dbea0e9c0d6971c.png"},{"id":32533339,"identity":"74aa1637-a6bf-47c8-a654-97b4f92c8877","added_by":"auto","created_at":"2023-02-06 14:46:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":641549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of oxamate on the expression of GLUT1 and GLUT3 in cartilage tissues after ACLT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) GLUT1 and (C) GLUT3 immunohistochemistry in knee joint sections from sham, ACLT, and ACLT + oxamate (0.25 mg/kg or 2.5 mg/kg) groups. The quantitative analysis of the ratio of (C) GLUT1-positive and (D) GLUT3-positive cells in joint sections are presented. Data are expressed as means ± SEM for each group. The scale bar represents 100 μm. (* p \u0026lt; 0.05, compared with the sham group; # p \u0026lt; 0.05, compared with the ACLT group).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2540780/v1/87f0ce95bd6a2dd89037ffa4.png"},{"id":32532330,"identity":"7cdb3923-a2ed-4b0c-9662-36e859e5964b","added_by":"auto","created_at":"2023-02-06 14:38:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":701991,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of oxamate on the expression of HK-II and PKM2 in cartilage tissues after ACLT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) HK-II and (C) PKM2 immunohistochemistry in joint sections from sham, ACLT, and ACLT + oxamate (0.25 mg/kg or 2.5 mg/kg) groups. The quantitative analysis of the ratio of (B) HK-II-positive and (D) PKM2-positive cells in joint sections are presented. Data are expressed as means ± SEM for each group. The scale bar represents 100 μm. (* p \u0026lt; 0.05, compared with the sham group; # p \u0026lt; 0.05, compared with the ACLT group).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2540780/v1/8a9239c72c187e4548f26d4b.png"},{"id":32532335,"identity":"e341468d-b9fc-4a2d-b66c-1c92bdba3048","added_by":"auto","created_at":"2023-02-06 14:38:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":392977,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of oxamate on the expression of LDHA in cartilage tissues after ACLT administration \u003c/strong\u003e(A) LDHA immunohistochemistry in joint sections from sham, ACLT, and ACLT + oxamate (0.25 mg/kg or 2.5 mg/kg) groups. (B) A quantitative analysis of the ratio of LDHA-positive cells in joint sections is presented. Data are expressed as means ± SEM for each group. The scale bar represents 100 μm. (* p \u0026lt; 0.05, compared with the sham group; # p \u0026lt; 0.05, compared with the ACLT group).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2540780/v1/cabb3a42afea15c344d2dcc4.png"},{"id":32532336,"identity":"15a1f085-db36-4329-b7e6-fae7a264c358","added_by":"auto","created_at":"2023-02-06 14:38:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":639928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of oxamate on the expression of PDK1 and PDK2 in cartilage tissues after ACLT administration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) PDK1 and (C) PDK2 immunohistochemistry in joint sections from sham, ACLT, and ACLT + oxamate (0.25 mg/kg or 2.5 mg/kg) groups. The quantitative analysis of the ratio of (B) PDK1-positive and (D) PDK2-positive cells in joint sections are presented. Data are expressed as means ± SEM for each group. The scale bar represents 100 μm. (* p \u0026lt; 0.05, compared with the sham group; # p \u0026lt; 0.05 , compared with the ACLT group).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2540780/v1/294aeff93bb2bd242cebd03c.png"},{"id":32534404,"identity":"e05330be-e0dc-4fc4-a36b-423825cbe7d1","added_by":"auto","created_at":"2023-02-06 14:54:56","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":300626,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of oxamate on the expression of TUNEL in cartilage tissues after ACLT administration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) TUNEL immunohistochemistry in joint sections from sham, ACLT, and ACLT + oxamate (0.25 mg/kg or 2.5 mg/kg) groups. (B) A quantitative analysis of the ratio of TUNEL-positive cells in joint sections is presented. Data are expressed as means ± SEM for each group. The scale bar represents 100 μm. (* p \u0026lt; 0.05, compared with sham group; # p \u0026lt; 0.05, compare with ACLT group).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-2540780/v1/5215c1505baa288754978d6b.png"},{"id":34882521,"identity":"e119d58d-cd99-43f8-b986-6deab47ae6c9","added_by":"auto","created_at":"2023-03-27 20:44:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4705159,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2540780/v1/d8d2055b-dfff-4ab2-bbd6-531ea46f5db9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The effects of intra-articular oxamate on anterior cruciate ligament transection-induced experimental osteoarthritic rats","fulltext":[{"header":"Background","content":"\u003cp\u003eSeveral complex mechanisms induce osteoarthritis (OA), including the progressive erosion of articular cartilage, proteoglycan degradation, and disruption of the collagen network, which lead to the progressive destruction of joints and loss of function [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Previous studies showed that metabolic pathways require reprogramming to adapt to energy changes following inflammatory responses [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. During the inflammation, cells preferentially use glycolysis to generate energy. After inflammation has resolved, oxidative phosphorylation (OXPHOS) is resumed to provide energy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eChondrocytes are the only cell type in cartilage and are mainly responsible for regulating extracellular matrix (ECM) repair and synthesis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. As cartilage tissues are avascular, nutrients and oxygen must be diffused from synovial fluid and subchondral bone to the chondrocytes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Chondrocyte metabolism occurs in a hypoxic environment, with glycolysis the major method of energy production [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Glucose transported to chondrocytes is converted to two pyruvate molecules and two ATP molecules in the glycolytic pathway. The glycolytic intermediate pyruvate can enter the tricarboxylic acid cycle (TCA cycle) via the pyruvate dehydrogenase (PDH) complex to generate 36 ATP molecules by mitochondrial OXPHOS. When changes in the cartilage tissue microenvironment occur, chondrocytes regulate metabolic pathways to adapt to environmental changes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn normal chondrocytes, around 25% of ATP originates from mitochondrial OXPHOS. However, as OA is accompanied by mitochondria dysfunctions in the cartilage, OXPHOS capacity decreases, resulting in energy deficiency [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Additionally, OA causes changes in energy metabolism pathways in chondrocytes, which are characterized by enhanced glycolysis and decreased OXPHOS capacity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Glycolysis-related proteins or enzymes, such as glucose transporters 1 (GLUT1) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], pyruvate kinase M2 (PKM2) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and lactate dehydrogenase A (LDHA) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], were reportedly upregulated in chondrocytes from patients with OA compared to healthy controls. The glycolytic intermediate, pyruvate, is converted into lactate by LDHA. Lactate, once considered an end product of glycolysis, has recently emerged as a critical regulator of OA development [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The inhibition of LDHA activity in chondrocytes by genetic manipulation (LDHA knockout) in mice, or pharmacological manipulation in vitro, has identified LDHA as a potential therapeutic target for OA treatment [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOxamate is a pyruvate analogue that mainly acts as a competitive inhibitor of LDHA to block the conversion of pyruvate to lactate [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Studies in recent years have reported the correlations between glycolysis and various diseases, including neurodegenerative diseases [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], rheumatoid arthritis (RA) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], cancer [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and diabetes [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Oxamate can inhibit glycolysis while stimulating mitochondrial OXPHOS to promote differentiation in mouse osteoprogenitors and human bone marrow stromal cells (BMSCs) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, to our knowledge, no other studies have examined the effects of oxamate in the treatment of OA. The present study used LDHA inhibitors, oxamate to elucidate the role of glycolysis-related enzymes on ACLT-induced OA in rats.\u003c/p\u003e"},{"header":"Mathods","content":"\u003cp\u003eAnimals and surgical technique for OA induction\u003c/p\u003e \u003cp\u003eOA was induced in male Wistar rats (8 weeks old, body weight 260\u0026ndash;280g) by anterior cruciate ligament transaction (ACLT) of the right knee; surgery was not performed on the left knee. This procedure was modified from the protocol described previously [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Each animal was administered a subcutaneous pre-operative injection of enrofloxacin (5 mg/kg) for prophylaxis. The animals were not immobilized after surgery and were allowed unrestricted cage activity. All rats were maintained in climate-controlled conditions on a 12-h light-dark cycle at 22\u0026ndash;24\u0026deg;C at a relative humidity of 50\u0026ndash;55%.\u003c/p\u003e \u003cp\u003eExperimental design and oxamate treatment\u003c/p\u003e \u003cp\u003eThe rats were randomly allocated into the following four experimental groups: Group I: sham; rats received arthrotomy without ACLT and were administered 50 \u0026micro;l of physiologically normal saline by intra-articular administration. Group II: ACLT; animals that underwent ACLT and were administered 50 \u0026micro;l of physiologically normal saline by intra-articular administration. Group III: ACLT\u0026thinsp;+\u0026thinsp;0.25 mg/kg oxamate; animals that underwent ACLT and were administered 0.25 mg/kg of oxamate (Thermo Fisher Scientific, Waltham, USA) by intra-articular administration once weekly for five consecutive weeks, beginning 10 weeks after surgery. Group IV: ACLT\u0026thinsp;+\u0026thinsp;2.5 mg/kg oxamate: animals that underwent ACLT and were administered 0.25 mg/kg of oxamate by intra-articular administration once weekly for five consecutive weeks, beginning 10 weeks after surgery. Ultimately, all animals were sacrificed, and their knee joints were collected for histopathological and immunohistochemical analyses. In our preliminary observation, compared with the ACLT group, changes in the hind paw weight-bearing distribution had decreased significantly, but the knee joint swelling had decreased insignificantly in ACLT\u0026thinsp;+\u0026thinsp;oxamate 0.125 mg/kg (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3). Therefore, we examine the effects of oxamate 0.25mg/kg and 2.5mg/kg on ACLT-rats.\u003c/p\u003e \u003cp\u003eTest for Defects in Hind Limb Weight-bearing and Joint Width Measurement\u003c/p\u003e \u003cp\u003eThe effect of joint damage on the weight distribution in OA and contra-lateral knees was measured using an incapacitance meter tester (Singa Technology Corporation, Taipei, TW) that independently measured the weight bearing of each hind paw. Changes in the distribution of hind paw weight-bearing were used as an index of joint discomfort as described previously [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Briefly, changes in hind paw weight-bearing distribution between the contralateral control and OA limbs were used as an index of joint pain in the surgically induced OA knee. The rats were placed in an angled Plexiglass chamber positioned so that each hind paw rested on a separate force plate. The force exerted by each hind limb (measured in grams) was averaged over a 5-s period. Each data point was the mean of three, 5-s readings. The hind paw weight distribution was expressed as the differences in weight between the contralateral and ipsilateral limbs. The width of the knee joint was measured from the medial to the lateral aspects of the knee joint at approximately the level of the medial and lateral joint lines using a vernier caliper (AA847R, Aesculap, AG\u0026amp;CO, KG, Osnabr\u0026uuml;ck, Germany). Changes in knee joint width, a measure of knee joint inflammation, were recorded weekly before and after ACLT for up to 26 weeks.\u003c/p\u003e \u003cp\u003eGross morphology and histopathological examination\u003c/p\u003e \u003cp\u003eAt week 26 after ACLT, the rats were sacrificed by deep anesthesia with sodium pentobarbital (50 mg/kg), then perfused intracardially with heparinized saline (200 ml/rat) followed by freshly prepared 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline, pH 7.4. The joints were sectioned 1 cm above and below the joint line, fixed in 10% neutral buffered formalin for 3 days, and then decalcified for 8 weeks in a buffered 4% ethylenediaminetetraacetic acid (EDTA) and formalin solution. The joints were then sectioned mid-sagittally, washed under running tap water, and paraffin-embedded using an automatic processor (Autotechnicon Mono 2; Technion Co., Chauncey, NY). Serial articular cartilage sections (1 \u0026micro;m) were cut on a rotatory microtome Microm HM340E (Walldorf, Germany) from the central weight-bearing surface of the femoral condyles and tibial plateau of both knees. Safranin-O/fast green staining was performed to assess the general morphology and matrix proteoglycan. Immediately after sacrifice, each knee was examined for gross morphologic changes in the cartilage lesions as described previously [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The articular cartilage was graded by microscopy according to the Osteoarthritis Research Society International (OARSI) system [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This system comprises six histological grades and four histological stages. The total score (score\u0026thinsp;=\u0026thinsp;grade \u0026times; stage) ranges from 1 point (normal articular cartilage) to 24 points (no repair).\u003c/p\u003e \u003cp\u003eImmunohistochemistry\u003c/p\u003e \u003cp\u003eCartilage specimens were processed for immunohistochemical analysis as described previously[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Briefly, 1 \u0026micro;m sections of paraffin-embedded specimens were placed on slides, deparaffinized with xylene, and dehydrated in an alcohol gradient. The antigen was retrieved by enzymatic digestion with proteinase K (20 mM) in Tris-EDTA buffer for 45 mins. The endogenous peroxidase activity then was quenched by 8-min incubation in 3% hydrogen peroxide. After washing three times for 8 mins in Tween-tris-buffered saline (TTBS), the sections were incubated in phosphate-buffered saline (PBS) containing 4% normal horse serum for 60 mines as a blocking agent for non-specific binding. The sections were incubated for 90 mines with biotinylated anti-rabbit or -mouse IgG (Vector Labs, Burlingame, CA) diluted 200-fold in 2% bovine serum albumin (BSA) in PBS. The sections were then treated using the avidin-biotin complex technique using an ABC kit (Vectastain A.B.C. kit; Vector Labs, Burlingame, CA). The images were viewed using a Leica DM6000 microscope (Leica, Heidelberg, Germany) and captured using a FLEXACAM C1 microscope camera (Leica, Heidelberg, Germany) The different antigens present in each cartilage specimens were quantified and estimated by determining the number of positively stained chondrocytes in the entire thickness of the cartilage, as described previously [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The cartilage was divided into six microscopic fields (three each in the superficial and deep zones) (magnification, 400\u0026times;), and the results were averaged. For each OA specimen, before evaluation, the presence of an intact cartilage surface that could be detected and used as a marker for the morphometric analyses. The data was expressed as the percentage of chondrocytes showing positive staining for the antigen (cell score), with the maximum cartilage specimen scores of 100%. Each slide was reviewed by two independent readers blinded to the treatment groups. The data obtained from the medial and lateral femoral condyle and tibial plateau were considered together for the statistical analyses.\u003c/p\u003e \u003cp\u003eData and statistical analysis\u003c/p\u003e \u003cp\u003eAll continuous data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used to test differences among the means of various scores in the experimental groups and for scores with significant differences. To compare mean differences between treatment and sham groups, Student-Newman-Keuls post hoc tests were used. The trends in the changes in nociceptive behavior and knee joint width were tested using repeated-measures ANOVA. Differences with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant, including the four groups in the present study.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eEffects of intra-articular oxamate on ACLT-induced nociception\u003c/p\u003e \u003cp\u003eIn order to assess the effects of oxamate on OA-induced weight-bearing changes and knee swelling, oxamate in 0.25 mg/kg and 2.5 mg/kg concentrations was administered via intraarticular (IA) injection once a week from Week 10 to Week 14 after ACLT surgery. In addition to the pain and joint instability caused by uneven weight bearing, rats with osteoarthritis (OA) experience an inflammatory response that includes joint swelling. Like the animal in the ACLT-group the animals in the ACLT\u0026thinsp;+\u0026thinsp;oxamate group showed no obvious neurological defects after ia oxamate injection. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, After ACLT surgery, weight-bearing changes increased in rats with time until before oxamate treatment in Week 10. The hind limbs of rats in the ACLT group (42.08\u0026thinsp;\u0026plusmn;\u0026thinsp;2.40 g) were shown to have significantly greater weight-bearing changes than those in the sham group (1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75 g). The comparison of the ACLT\u0026thinsp;+\u0026thinsp;oxamate 0.25 mg/kg group and the ACLT group found that differences after Week 16 were significant and persisted until Week 26. The comparison of the ACLT\u0026thinsp;+\u0026thinsp;oxamate 2.5 mg/kg group and the ACLT group found that differences after Week 16 were significant and persisted until Week 26. Based on the above results, oxamate at 0.25 mg/kg, and 2.5 mg/kg can effectively improve lower-limb weight-bearing distribution in the ACLT-induced OA model. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, after ACLT surgery, knee joint swelling increased in rats with time until before oxamate treatment in Week 10. Rats in the ACLT group (0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 mm) exhibited significantly higher knee joint swelling than those in the sham group (0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mm). The comparison of the ACLT\u0026thinsp;+\u0026thinsp;oxamate 0.25 mg/kg treatment group and the ACLT group found that differences after Week 16 were significant and persisted until Week 26. The comparison of the ACLT\u0026thinsp;+\u0026thinsp;oxamate 2.5mg/kg group and the ACLT group found that differences after Week 14 were significant and persisted until Week 26. Based on the above results, oxamate 0.125 mg/kg, 0.25mg/kg, and 2.5mg/kg can effectively improve knee joint swelling in the ACLT-induced OA model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOxamate attenuates cartilage degradation in ACLT-rats\u003c/p\u003e \u003cp\u003eSafranin O/Fast Green staining was used to assess the protective effects of oxamate in cartilage degradation/damage, and the Osteoarthritis Research Society International (OARSI) system was used to analyze knee joint tissues in the sham, ACLT, and ACLT\u0026thinsp;+\u0026thinsp;oxamate (0.25 mg/kg or 2.5mg/kg) groups. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. Compared to the sham group, extracellular matrix (ECM) loss and severe damage to the articular surface were observed in the ACLT group. Although ECM loss was not alleviated in the ACLT\u0026thinsp;+\u0026thinsp;oxamate 0.25 mg/kg group, there was an improvement in articular surface damage. ECM loss and articular surface injury were improved in the ACLT\u0026thinsp;+\u0026thinsp;oxamate 2.5mg/kg group. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB shows the quantitative analysis results by the OARSI system: The ACLT group (8.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73) scored significantly higher than the sham group (0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33). Although the ACLT\u0026thinsp;+\u0026thinsp;oxamate 0.25 mg/kg group (4.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.83, p\u0026thinsp;=\u0026thinsp;0.205) had a lower OARSI score than the ACLT group, the difference was insignificant. The ACLT\u0026thinsp;+\u0026thinsp;oxamate 2.5 mg/kg group (2.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73) had a significantly lower OARSI score than the ACLT group. Based on the above results, treatment with oxamate at 2.5 mg/kg can significantly reduce cartilage ECM and articular surface injury in the ACLT-induced OA model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOxamate affects glucose-transporter 1 (GLUT1) and GLUT3 expression in ACLT cartilage\u003c/p\u003e \u003cp\u003eGLUT1 and GLUT3 are GLUT isoforms, and their expression indicates an increased glucose uptake, thereby promoting glycolysis. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. The ACLT group exhibited increased GLUT1 protein expression in cartilage tissues (above the dotted line) compared to the sham group. Treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg can significantly decrease GLUT1 protein expression in ACLT-cartilage tissues. The quantitative results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB. GLUT1 protein expression was significantly higher in the ACLT group than in the sham group. After treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg, GLUT1 protein expression was significantly downregulated compared to that in the ACLT group. The above results showed that oxamate at 0.25 mg/kg and 2.5 mg/kg can decrease GLUT1 protein expression in ACLT-cartilage tissues. The results of GLUT3 protein expression are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC. Compared to the sham group, GLUT3 protein expression was increased in cartilage tissues (above the dotted line) in the ACLT group. Treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg can significantly decrease GLUT3 protein expression in ACLT-cartilage tissues. The quantitative results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD. GLUT3 protein expression was significantly higher in the ACLT group than in the sham group. After treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg, GLUT3 protein expression was significantly downregulated compared to that in the ACLT group. The above results showed that oxamate at 0.25 mg/kg and 2.5 mg/kg can decrease GLUT3 protein expression in ACLT-cartilage tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOxamate affects hexokinase II (HK-II) expression in ACLT cartilage\u003c/p\u003e \u003cp\u003eHK-II is a major regulatory protein in glycolysis, and its expression can increase the conversion rate of glucose to glucose 6-phosphate (G6P), thereby promoting glycolysis. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. Compared to the sham group, HK-II protein expression was increased in cartilage tissues (above the dotted line) in the ACLT group.Treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg can decrease HK-II protein expression in ACLT-cartilage tissues. The quantitative results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. HK-II protein expression was significantly higher in the ACLT group than in the sham group. Although HK-II protein expression was downregulated after treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg compared to that in the ACLT group, the decreases were not significant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOxamate affects pyruvate kinase M2 (PKM2) expression in ACLT cartilage\u003c/p\u003e \u003cp\u003ePKM2 is a PK isoform that helps cells to obtain energy via glycolysis in a hypoxic environment. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC. Compared to the same group, PKM2 protein expression was increased in cartilage tissues (above the dotted line) in the ACLT group.Treatment with oxamate at 2.5 mg/kg can decrease PKM2 protein expression in ACLT-cartilage. The quantitative results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD. Compared to the sham group, PKM2 protein expression was significantly increased in the ACLT group. Although PKM2 protein expression was downregulated after treatment with oxamate at 0.25 mg/kg compared to that in the ACLT group (p\u0026thinsp;=\u0026thinsp;0.318), the decrease was not significant. PKM2 protein expression was significantly downregulated after treatment with oxamate at 2.5 mg/kg compared to the ACLT group. Based on the above results, treatment with oxamate at 2.5 mg/kg can significantly decrease PKM2 protein expression in cartilage tissues in the ACLT-induced OA model.\u003c/p\u003e \u003cp\u003eOxamate affects lactate dehydrogenase A (LDHA) expression in ACLT cartilage\u003c/p\u003e \u003cp\u003eLDHA can convert pyruvate synthesized during glycolysis to lactate in a hypoxic environment. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. The ACLT group exhibited increased LDHA protein expression in cartilage tissues (above the dotted line) compared to the sham group. Treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg can decrease LDHA protein expression in ACLT-cartilage tissues. The quantitative results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. LDHA protein expression was significantly higher in the ACLT group than in the sham group. After treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg, LDHA protein expression was significantly downregulated compared to that in the ACLT group. The above results showed that oxamate at 0.25 mg/kg and 2.5 mg/kg can decrease LDHA protein expression in ACLT-cartilage tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOxamate affects pyruvate dehydrogenase kinase 1 (PDK1) and PDK2 expression in ACLT cartilage\u003c/p\u003e \u003cp\u003ePDK1 and PDK2 are PDK isoforms with the primary function of inhibiting acetyl-CoA entry into the tricarboxylic acid cycle and decreasing OXPHOS. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. Compared to the sham group, PDK1 protein expression was increased in cartilage tissues (above the dotted line) in the ACLT group. Treatment with oxamate at 2.5 mg/kg can decrease PDK1 protein expression in ACLT-cartilage tissues. The quantitative results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB. Compared to the sham group, PDK1 protein expression was significantly increased in the ACLT group. Although PDK1 protein expression was downregulated after treatment with oxamate at 0.25 mg/kg compared to that in the ACLT group (p\u0026thinsp;=\u0026thinsp;0.171), the decrease was not significant. PDK1 protein expression was significantly downregulated after treatment with oxamate at 2.5 mg/kg compared to that in the ACLT group. Based on the above results, treatment with oxamate at 2.5 mg/kg can significantly decrease PDK1 protein expression in cartilage tissues in the ACLT-induced OA model. The results of PDK2 protein expression are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. Compared to the sham group, PDK2 protein expression was increased in cartilage tissues (above the dotted line) in the ACLT group. Treatment with oxamate at 0.25 mg/kg and 2.5mg/kg can decrease PDK2 protein expression in ACLT-cartilage tissues. The quantitative results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB. Compared to the sham group, PDK2 protein expression was significantly increased in the ACLT group. After treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg, PDK2 protein expression was significantly downregulated compared to that in the ACLT group. The above results showed that oxamate at 0.25 mg/kg and 2.5 mg/kg can decrease PDK2 protein expression in ACLT-cartilage tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOxamate affects TUNEL expression in ACLT cartilage\u003c/p\u003e \u003cp\u003eThe results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA. The ACLT group exhibited increased TUNEL expression in cartilage tissues (above the dotted line) compared to the sham group. Treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg can significantly decrease TUNEL protein expression in ACLT-cartilage tissues. The quantitative results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB. TUNEL expression was significantly higher in the ACLT group than in the sham group. After treatment with oxamate at 0.25 mg/kg and 2.5 mg/kg, TUNEL protein expression was significantly downregulated compared to that in the ACLT group. The above results showed that oxamate at 0.25 mg/kg and 2.5 mg/kg can decrease TUNEL protein expression in ACLT-cartilage tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBecause articular cartilage lacks vascular, neural, and lymphatic tissues, nutrients and oxygen are transported by diffusion from synovial fluid secreted by synovial tissues. Oxamate is an LDHA inhibitor, and previous research has shown that it mainly regulates glycolysis when used for inhibiting cancer cell growth [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The present results indicated that IA administering oxamate to rats with ACLT-induced OA could significantly improve weight-bearing defects and knee joint swelling in their hind limbs. From histopathological observations, oxamate inhibits ACLT-induced cartilage degradation and chondrocyte apoptosis. At the same time, oxamate decreases the upregulation of glycolysis-related proteins such as GLUT1, GLUT3, HK-II, PKM2, LDHA, PDK1, and PDK2 in cartilage tissues in ACLT-induced OA.\u003c/p\u003e \u003cp\u003eBased on clinical study, there is a 50% chance of developing knee OA 10\u0026ndash;20 years after ACL injury [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Histopathological examination conducted in rat experiments revealed that OA induced by this surgery is caused by joint instability combined with intense pain, resulting in uneven weight bearing in the hind limbs and articular cartilage degeneration [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Several studies have demonstrated that the progression of OA is accompanied by nociceptive behaviors [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Previous in vivo studies also have indicated that the ACLT-induced OA model allows clear observation of mechanical allodynia and changes in Weight-bearing distribution[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Moreover, there will be swelling accompanying the injured knee joint with disease progression after ACLT [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The present study found that an IA injection of oxamate after surgery could attenuate pain behavior due to weight-bearing defects in the hind limb (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In addition to pain and joint instability, patients with OA exhibit inflammatory responses that include joint swelling and synovitis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Previous studies have suggested that ACLT surgery induces early OA in rats, and its joint pathology increases joint diameter, cartilage degeneration, synovitis, and chondrocyte death [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The results of this study were similar to previous studies: There was significant knee joint swelling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and cartilage degeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) in the ACLT group, and an IA injection with oxamate could significantly improve the aforementioned pathological phenomenon.\u003c/p\u003e \u003cp\u003eUp to the present, GLUTs have 14 isoforms [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Their function is to transport extracellular glucose into cells, thereby providing a source of glucose for glycolysis. Studies have found that GLUT1, GLUT3, and GLUT9 are expressed in normal chondrocytes, with GLUT1 and GLUT3 being extremely sensitive to hypoxic environments [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In 2013, V\u0026aacute;zquez-Mosquera et al. proved that GLUT1 and GLUT3 expressions in cartilage tissues in patients with OA were higher than in healthy people, but these differences were insignificant [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. This study showed that GLUT1 and GLUT3 expressions in cartilage tissue were significantly upregulated in the ACLT-induced OA group compared to those in the na\u0026iuml;ve group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Previous research found that while increasing glucose transport helps cells obtain energy, it also causes oxidative stress and inflammation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Upregulation of oxidative stress and inflammation can damage chondrocytes and ultimately exacerbate OA [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. According to our findings, an IA injection of oxamate inhibits ACLT-induced GLUT1 and GLUT3 expressions in chondrocytes (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). We hypothesize that oxamate downregulates the expression of these 2 GLUTs, which may regulate more efficient energy generation methods (discussed after this section) and reduce oxidative stress and inflammation, both of which have protective effects in OA.\u003c/p\u003e \u003cp\u003eIn an anaerobic phase, chondrocytes in OA synthesize pyruvate via glycolysis and LDHA converts pyruvate to lactate. This conversion pathway can generate two ATP molecules to provide energy, but lactate accumulation causes an acidic microenvironment [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Studies have shown that low extracellular pH can regulate the expression of ECM-degrading enzymes. Moreover, extracellular pH\u0026thinsp;\u0026lt;\u0026thinsp;7.1 inhibits ECM synthesis while increasing the expression of ECM-degrading enzymes, resulting in cartilage degeneration [\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In addition, the acidic environment in the OA articular cavity causes pain [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In 2020, Arra et al. used an \u003cem\u003eex vivo\u003c/em\u003e OA model to demonstrate that inflammation causes chondrocyte glycolysis, elevates LDHA, and decreases OXPHOS [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Downregulation of LDHA can reduce the expression of the ECM-degrading enzyme MMP13 and protects cartilage tissues in OA animal experiments [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Our findings also showed that in ACLT-induced OA rats, LDHA protein expression was upregulated in chondrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), which was accompanied by ECM loss and surface damage in cartilage tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Following IA administration with the LHDA inhibitor (oxamate), ECM loss and cartilage surface injury were significantly improved. Simultaneously, IHC staining results revealed that an IA injection of oxamate could significantly inhibit LDHA protein expression in ACLT-induced OA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Previous reports and the present results found that LHDA inhibition and downregulated protein expression protect joints in OA.\u003c/p\u003e \u003cp\u003eNormal chondrocytes receive 75% of their energy from glycolysis, but mitochondria receive 25% of their energy from OXPHOS [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. An oxygen gradient microenvironment exists in cartilage tissues, and chondrocytes in the deepest part can obtain 1% oxygen. Hence, both glycolysis and OXPHOS occur in chondrocytes [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. A study revealed that most chondrocytes\u0026rsquo; energy demands are met by glycolysis rather than OXPHOS [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Although OXPHOS provides 25% of ATP to chondrocytes, this pathway can synthesize 36 ATP molecules from a single glucose molecule and its energy generation efficiency is far greater than glycolysis, which can only synthesize 2 ATP molecules from 1 glucose molecule [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In 2015, Qu et al. demonstrated that while glycolysis was increased in OA chondrocytes, ATP energy acquisition was decreased, inhibiting chondrocyte proliferation, differentiation, and cell viability [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. We used oxamate to inhibit OA-induced LHDA protein upregulation. This might allow more intermediate product of glycolysis, pyruvate to enter mitochondrial OXPHOS and produce more energy for repairing damaged chondrocytes and decreasing apoptosis. Therefore, oxamate can improve cartilage degeneration and chondrocyte death.\u003c/p\u003e \u003cp\u003eThe pyruvate dehydrogenase complex (PDC) is essential in aerobic metabolism because it catalyzes pyruvate entry into mitochondria for OXPHOS [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. PDC activity is negatively regulated by PDK. In pathological conditions (e.g., diabetes, cancer, and sepsis), PDK activation inhibits PDC activity and prevents cytoplasmic pyruvate from entering mitochondria for OXPHOS, resulting in decreased ATP synthesis [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The previous study has found that PDK decreases PDC activity, it increases the glucose metabolites pyruvate and lactate while decreasing mitochondrial OXPHOS [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Our experiments found that ACLT upregulated PDK1 and PDK2 in chondrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), which may inhibit PDC activity, thereby affecting pyruvate entry into the mitochondrial OXPHOS for ATP synthesis. The previous study also indicate that a hypoxic environment can increases hypoxia-inducible factor 1-alpha (HIF-1α) expression and activates PDK1 [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. This results in the reprogramming of glucose metabolism from OXPHOS to aerobic glycolysis and a decrease in ATP synthesis [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Yudoh et al. (2004) demonstrated that HIF-1α expression is elevated in the cartilage tissues of patients with OA and contributes to cartilage degeneration [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Therefore, we believe that ACLT-induced PDK upregulation is caused by HIF-1α.\u003c/p\u003e \u003cp\u003eIts primary function of HK is to convert phosphorylated glucose to glucose 6-phosphate [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. HK-IIcan reprogram tumor cells\u0026rsquo; metabolisms to aerobic glycolysis [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. A study revealed that HK-II expression in peripheral blood mononuclear cells is higher in patients with OA than in healthy people [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. This study found that ACLT upregulates HK-II expression in chondrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Many studies have proved that transforming growth factor beta (TGF-β) plays an important role in OA progression [\u003cspan additionalcitationids=\"CR70\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. A previous study found that TGF-β1 upregulates HK-II protein expression in OA chondrocytes and induces aerobic glycolysis, increases glucose consumption and lactate synthesis, and decreases ATP synthesis simultaneously [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Our previous studies also found that ACLT increases TGF-β1 in chondrocytes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, we suggest that ACLT-induced HK-II upregulation may be regulated by TGF-β.\u003c/p\u003e \u003cp\u003ePyruvate kinase(PK) has two isoforms (PKM1 and PKM2) and mainly mediates the final step in glycolysis, which converts phosphoenolpyruvate to pyruvate, and this step generates one ATP [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Previous studies proved that PKM2 expression in the cartilage of patients with OA was higher than that of healthy people and that PKM2 knockdown could downregulate GLUT1, HIF-1α, and LDHA expressions, inhibit OA chondrocyte proliferation, and promote apoptosis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The present study found that the PKM2 protein is significantly increased in chondrocytes when OA occurs and that oxamate injection significantly downregulates the expression of this protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). This could be because oxamate inhibits LDHA, PDK1 and PDK2 expressions in the ACLT group while partially restoring ATP synthesis in the OXPHOS pathway.\u003c/p\u003e \u003cp\u003ePrevious studies have proved that oxamate can promote bone formation and bone strength in mice [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], but its protective mechanisms in OA are unknown. This study found that an IA injection of oxamate inhibits ACLT-induced LDHA, PDK1, and PDK2 upregulation and may promote OXPHOS restoration to increase ATP synthesis, assist in chondrocyte repair, and decrease apoptosis. However, as long as ATP supply is available in sufficient quantities, glycolysis-related proteins such as GLUT1, GLUT3, HK-II, and PKM2 may be indirectly regulated by oxamate in ACLT-induced OA.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe results of this study support the fact that oxamate can regulate the expression of glucose metabolism proteins and partially restore OXPHOS to provide more ATP to protect chondrocytes. We believe that an IA injection of oxamate has protective effects in OA and has potential for development.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eACLT\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAnterior cruciate ligament transection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eECM\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eGLUT1\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlucose-transporter 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eGLUT3\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlucose-transporter 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eHK-II\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHexokinase II\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eIA\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIntraarticular\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eLDHA\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLactate dehydrogenase A\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eOA\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOsteoarthritis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eOXPHOS\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOxidative phosphorylation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePDC\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePyruvate dehydrogenase complex\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePDH\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePyruvate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePDK1\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePyruvate dehydrogenase kinase 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePDK2\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePyruvate dehydrogenase kinase 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePK\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePyruvate kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePKM2\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePyruvate kinase M2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eTCA cycle\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTricarboxylic acid cycle\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eTUNEL\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTerminal deoxynucleotidyl transferase dutp nick-end labeling\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe animal experiments were performed according to the Guiding Principles in the Care and Use of Animals, as approved by the Council of the American Physiology Society, and were approved by the National Sun Yat-sen University Animal Care and Use Committee (approval no.11028).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe datasets supporting the conclusions of this study are included within the article.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe study was supported by the National Science and Technology Council, Taiwan (NSCT 110-2314-B-843-001; NSCT 111-2314-B-843-001; NSCT 109-2314-B-075) and partly supported by Ping-Tung Christian Hospital.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eZhi-Hong Wen: Conceptualization, Writing-Original draft preparation and Formal analysis; Chun-Sung Sung:\u0026nbsp;Conceptualization, Writing-Reviewing and Editing, \u0026nbsp;Formal analysis and Funding acquisition; Yen-Hsuan Jean: Conceptualization, Writing-Original draft preparation, Formal analysis and Funding acquisition;\u0026nbsp;Sung-Chun Lin: Writing-Reviewing and Editing; Zhi-Kang Yao: Software and Validation;\u0026nbsp;Yu-Wei Liu: Methodology and Investigation; Yu-Yan Wu: Methodology and Investigation;\u0026nbsp;Yu-Cheng Lai: Software, Validation and Formal analysis; Wu-Fu Chen: Writing- Reviewing and Editing. Hsin-Tzu Liu: Visualization.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe authors also thank editage (www.editage.com.tw) for the English language review.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWalsh MC, Kim N, Kadono Y, Rho J, Lee SY, Lorenzo J, Choi Y: \u003cb\u003eOSTEOIMMUNOLOGY: Interplay Between the Immune System and Bone Metabolism\u003c/b\u003e. Annu Rev Immunol 2006, \u003cb\u003e24\u003c/b\u003e(1):33\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu C, Xuan Y, Zhang X, Liu Y, Yang S, Yang K: \u003cb\u003eImmune cell metabolism and metabolic reprogramming\u003c/b\u003e. Mol Biol Rep 2022, \u003cb\u003e49\u003c/b\u003e(10):9783\u0026ndash;9795.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoto-Heredero G, Gomez de Las Heras MM, Gabande-Rodriguez E, Oller J, Mittelbrunn M: \u003cb\u003eGlycolysis - a key player in the inflammatory response\u003c/b\u003e. FEBS J 2020, \u003cb\u003e287\u003c/b\u003e(16):3350\u0026ndash;3369.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkkiraju H, Nohe A: \u003cb\u003eRole of Chondrocytes in Cartilage Formation, Progression of Osteoarthritis and Cartilage Regeneration\u003c/b\u003e. J Dev Biol 2015, \u003cb\u003e3\u003c/b\u003e(4):177\u0026ndash;192.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLe Clanche S, Bonnefont-Rousselot D, Sari-Ali E, Rannou F, Borderie D: \u003cb\u003eInter-relations between osteoarthritis and metabolic syndrome: A common link?\u003c/b\u003e Biochimie 2016, \u003cb\u003e121\u003c/b\u003e:238\u0026ndash;252.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLane RS, Fu Y, Matsuzaki S, Kinter M, Humphries KM, Griffin TM: \u003cb\u003eMitochondrial respiration and redox coupling in articular chondrocytes\u003c/b\u003e. Arthritis Res Ther 2015, \u003cb\u003e17\u003c/b\u003e:54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishida T, Kubota S, Aoyama E, Takigawa M: \u003cb\u003eImpaired glycolytic metabolism causes chondrocyte hypertrophy-like changes via promotion of phospho-Smad1/5/8 translocation into nucleus\u003c/b\u003e. Osteoarthritis Cartilage 2013, \u003cb\u003e21\u003c/b\u003e(5):700\u0026ndash;709.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng L, Zhang Z, Sheng P, Mobasheri A: \u003cb\u003eThe role of metabolism in chondrocyte dysfunction and the progression of osteoarthritis\u003c/b\u003e. Ageing Research Reviews 2021, \u003cb\u003e66\u003c/b\u003e:101249.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu X, Fan X, Crawford R, Xiao Y, Prasadam I: \u003cb\u003eThe Metabolic Landscape in Osteoarthritis\u003c/b\u003e. Aging Dis 2022, \u003cb\u003e13\u003c/b\u003e(4):1166\u0026ndash;1182.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMobasheri A, Rayman MP, Gualillo O, Sellam J, van der Kraan P, Fearon U: \u003cb\u003eThe role of metabolism in the pathogenesis of osteoarthritis\u003c/b\u003e. Nature Reviews Rheumatology 2017, \u003cb\u003e13\u003c/b\u003e(5):302\u0026ndash;311.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKudelko M, Chan CW, Sharma R, Yao Q, Lau E, Chu IK, Cheah KS, Tanner JA, Chan D: \u003cb\u003eLabel-Free Quantitative Proteomics Reveals Survival Mechanisms Developed by Hypertrophic Chondrocytes under ER Stress\u003c/b\u003e. Journal of proteome research 2016, \u003cb\u003e15\u003c/b\u003e(1):86\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePfander D, Cramer T, Swoboda B: \u003cb\u003eHypoxia and HIF-1alpha in osteoarthritis\u003c/b\u003e. Int Orthop 2005, \u003cb\u003e29\u003c/b\u003e(1):6\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang X, Chen W, Zhao X, Chen L, Li W, Ran J, Wu L: \u003cb\u003ePyruvate Kinase M2 Modulates the Glycolysis of Chondrocyte and Extracellular Matrix in Osteoarthritis\u003c/b\u003e. DNA Cell Biol 2018, \u003cb\u003e37\u003c/b\u003e(3):271\u0026ndash;277.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArra M, Swarnkar G, Ke K, Otero JE, Ying J, Duan X, Maruyama T, Rai MF, O'Keefe RJ, Mbalaviele G \u003cem\u003eet al\u003c/em\u003e: \u003cb\u003eLDHA-mediated ROS generation in chondrocytes is a potential therapeutic target for osteoarthritis\u003c/b\u003e. Nat Commun 2020, \u003cb\u003e11\u003c/b\u003e(1):3427.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManeiro E, Martin MA, de Andres MC, Lopez-Armada MJ, Fernandez-Sueiro JL, del Hoyo P, Galdo F, Arenas J, Blanco FJ: \u003cb\u003eMitochondrial respiratory activity is altered in osteoarthritic human articular chondrocytes\u003c/b\u003e. Arthritis Rheum 2003, \u003cb\u003e48\u003c/b\u003e(3):700\u0026ndash;708.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRichardson SM, Hoyland JA, Mobasheri R, Csaki C, Shakibaei M, Mobasheri A: \u003cb\u003eMesenchymal stem cells in regenerative medicine: opportunities and challenges for articular cartilage and intervertebral disc tissue engineering\u003c/b\u003e. J Cell Physiol 2010, \u003cb\u003e222\u003c/b\u003e(1):23\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhai X, Yang Y, Wan J, Zhu R, Wu Y: \u003cb\u003eInhibition of LDH-A by oxamate induces G2/M arrest, apoptosis and increases radiosensitivity in nasopharyngeal carcinoma cells\u003c/b\u003e. Oncol Rep 2013, \u003cb\u003e30\u003c/b\u003e(6):2983\u0026ndash;2991.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang BLJJoCP: \u003cb\u003eGlucose\u003c/b\u003e, \u003cb\u003eglycolysis\u003c/b\u003e, \u003cb\u003eand neurodegenerative diseases\u003c/b\u003e. 2020, \u003cb\u003e235\u003c/b\u003e(11):7653\u0026ndash;7662.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbboud G, Choi S-C, Kanda N, Zeumer-Spataro L, Roopenian DC, Morel LJFii: \u003cb\u003eInhibition of glycolysis reduces disease severity in an autoimmune model of rheumatoid arthritis\u003c/b\u003e. 2018, \u003cb\u003e9\u003c/b\u003e:1973.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Ziaydi AG, Al-Shammari AM, Hamzah MI, Kadhim HS, Jabir MSJV: \u003cb\u003eNewcastle disease virus suppress glycolysis pathway and induce breast cancer cells death\u003c/b\u003e. 2020, \u003cb\u003e31\u003c/b\u003e(3):341\u0026ndash;348.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo X, Li H, Xu H, Woo S, Dong H, Lu F, Lange AJ, Wu C: \u003cb\u003eGlycolysis in the control of blood glucose homeostasis\u003c/b\u003e. Acta Pharmaceutica Sinica B 2012, \u003cb\u003e2\u003c/b\u003e(4):358\u0026ndash;367.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHollenberg AM, Smith CO, Shum LC, Awad H, Eliseev RA: \u003cb\u003eLactate Dehydrogenase Inhibition With Oxamate Exerts Bone Anabolic Effect\u003c/b\u003e. J Bone Miner Res 2020, \u003cb\u003e35\u003c/b\u003e(12):2432\u0026ndash;2443.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStoop R, Buma P, van der Kraan PM, Hollander AP, Billinghurst RC, Meijers TH, Poole AR, van den Berg WB: \u003cb\u003eType II collagen degradation in articular cartilage fibrillation after anterior cruciate ligament transection in rats\u003c/b\u003e. Osteoarthritis Cartilage 2001, \u003cb\u003e9\u003c/b\u003e(4):308\u0026ndash;315.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJean YH, Wen ZH, Chang YC, Hsieh SP, Tang CC, Wang YH, Wong CS: \u003cb\u003eIntra-articular injection of the cyclooxygenase-2 inhibitor parecoxib attenuates osteoarthritis progression in anterior cruciate ligament-transected knee in rats: role of excitatory amino acids\u003c/b\u003e. Osteoarthritis Cartilage 2007, \u003cb\u003e15\u003c/b\u003e(6):638\u0026ndash;645.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBove SE, Laemont KD, Brooker RM, Osborn MN, Sanchez BM, Guzman RE, Hook KE, Juneau PL, Connor JR, Kilgore KS: \u003cb\u003eSurgically induced osteoarthritis in the rat results in the development of both osteoarthritis-like joint pain and secondary hyperalgesia\u003c/b\u003e. Osteoarthritis and Cartilage 2006, \u003cb\u003e14\u003c/b\u003e(10):1041\u0026ndash;1048.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWen ZH, Lin YY, Chang YC, Tang CC, Hsieh SP, Lee HP, Sung CS, Chen WF, Lee CH, Hsuan Jean Y: \u003cb\u003eThe COX-2 inhibitor etoricoxib reduces experimental osteoarthritis and nociception in rats: The roles of TGF-beta1 and NGF expressions in chondrocytes\u003c/b\u003e. Eur J Pain 2020, \u003cb\u003e24\u003c/b\u003e(1):209\u0026ndash;222.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFernandes JC, Martel-Pelletier J, Otterness IG, Lopez-Anaya A, Mineau F, Tardif G, Pelletier JP: \u003cb\u003eEffects of tenidap on canine experimental osteoarthritis. I. Morphologic and metalloprotease analysis\u003c/b\u003e. Arthritis Rheum 1995, \u003cb\u003e38\u003c/b\u003e(9):1290\u0026ndash;1303.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePritzker KP, Gay S, Jimenez SA, Ostergaard K, Pelletier JP, Revell PA, Salter D, van den Berg WB: \u003cb\u003eOsteoarthritis cartilage histopathology: grading and staging\u003c/b\u003e. Osteoarthritis Cartilage 2006, \u003cb\u003e14\u003c/b\u003e(1):13\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWen ZH, Tang CC, Chang YC, Huang SY, Lin YY, Hsieh SP, Lee HP, Lin SC, Chen WF, Jean YH: \u003cb\u003eCalcitonin attenuates cartilage degeneration and nociception in an experimental rat model of osteoarthritis: role of TGF-beta in chondrocytes\u003c/b\u003e. Sci Rep 2016, \u003cb\u003e6\u003c/b\u003e:28862.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoileau C, Martel-Pelletier J, Brunet J, Schrier D, Flory C, Boily M, Pelletier JP: \u003cb\u003ePD-0200347, an alpha2delta ligand of the voltage gated calcium channel, inhibits in vivo activation of the Erk1/2 pathway in osteoarthritic chondrocytes: a PKCalpha dependent effect\u003c/b\u003e. Ann Rheum Dis 2006, \u003cb\u003e65\u003c/b\u003e(5):573\u0026ndash;580.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFiume L, Vettraino M, Manerba M, Di Stefano G: \u003cb\u003eInhibition of lactic dehydrogenase as a way to increase the anti-proliferative effect of multi-targeted kinase inhibitors\u003c/b\u003e. Pharmacol Res 2011, \u003cb\u003e63\u003c/b\u003e(4):328\u0026ndash;334.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Z, Han F, Yang S, Wu J, Zhan W: \u003cb\u003eOxamate-mediated inhibition of lactate dehydrogenase induces protective autophagy in gastric cancer cells: involvement of the Akt-mTOR signaling pathway\u003c/b\u003e. Cancer Lett 2015, \u003cb\u003e358\u003c/b\u003e(1):17\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLouboutin H, Debarge R, Richou J, Selmi TA, Donell ST, Neyret P, Dubrana F: \u003cb\u003eOsteoarthritis in patients with anterior cruciate ligament rupture: a review of risk factors\u003c/b\u003e. Knee 2009, \u003cb\u003e16\u003c/b\u003e(4):239\u0026ndash;244.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTawonsawatruk T, Sriwatananukulkit O, Himakhun W, Hemstapat W: \u003cb\u003eComparison of pain behaviour and osteoarthritis progression between anterior cruciate ligament transection and osteochondral injury in rat models\u003c/b\u003e. Bone Joint Res 2018, \u003cb\u003e7\u003c/b\u003e(3):244\u0026ndash;251.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLitwic A, Edwards MH, Dennison EM, Cooper C: \u003cb\u003eEpidemiology and burden of osteoarthritis\u003c/b\u003e. Br Med Bull 2013, \u003cb\u003e105\u003c/b\u003e:185\u0026ndash;199.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeogi T: \u003cb\u003eThe epidemiology and impact of pain in osteoarthritis\u003c/b\u003e. Osteoarthritis Cartilage 2013, \u003cb\u003e21\u003c/b\u003e(9):1145\u0026ndash;1153.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBartley EJ, Palit S, Staud R: \u003cb\u003ePredictors of Osteoarthritis Pain: the Importance of Resilience\u003c/b\u003e. Curr Rheumatol Rep 2017, \u003cb\u003e19\u003c/b\u003e(9):57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWen ZH, Tang CC, Chang YC, Huang SY, Chen CH, Wu SC, Hsieh SP, Hsieh CS, Wang KY, Lin SY \u003cem\u003eet al\u003c/em\u003e: \u003cb\u003eIntra-articular injection of the selective cyclooxygenase-2 inhibitor meloxicam (Mobic) reduces experimental osteoarthritis and nociception in rats\u003c/b\u003e. Osteoarthritis Cartilage 2013, \u003cb\u003e21\u003c/b\u003e(12):1976\u0026ndash;1986.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKao JH, Lin SH, Lai CF, Lin YC, Kong ZL, Wong CS: \u003cb\u003eShea Nut Oil Triterpene Concentrate Attenuates Knee Osteoarthritis Development in Rats: Evidence from Knee Joint Histology\u003c/b\u003e. PLoS One 2016, \u003cb\u003e11\u003c/b\u003e(9):e0162022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBijlsma JW, Berenbaum F, Lafeber FP: \u003cb\u003eOsteoarthritis: an update with relevance for clinical practice\u003c/b\u003e. Lancet 2011, \u003cb\u003e377\u003c/b\u003e(9783):2115\u0026ndash;2126.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarbosa GM, Cunha JE, Cunha TM, Martinho LB, Castro P, Oliveira FFB, Cunha FQ, Ramalho FS, Salvini TF: \u003cb\u003eClinical-like cryotherapy improves footprint patterns and reduces synovial inflammation in a rat model of post-traumatic knee osteoarthritis\u003c/b\u003e. Sci Rep 2019, \u003cb\u003e9\u003c/b\u003e(1):14518.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Li P, Zhu S, Bi R: \u003cb\u003eComparison of early-stage changes of osteoarthritis in cartilage and subchondral bone between two different rat models\u003c/b\u003e. PeerJ 2020, \u003cb\u003e8\u003c/b\u003e:e8934.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePickarski M, Hayami T, Zhuo Y, Duong LT: \u003cb\u003eMolecular changes in articular cartilage and subchondral bone in the rat anterior cruciate ligament transection and meniscectomized models of osteoarthritis\u003c/b\u003e. BMC Musculoskelet Disord 2011, \u003cb\u003e12\u003c/b\u003e:197.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNavale AM, Paranjape AN: \u003cb\u003eGlucose transporters: physiological and pathological roles\u003c/b\u003e. Biophys Rev 2016, \u003cb\u003e8\u003c/b\u003e(1):5\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMobasheri A, Neama G, Bell S, Richardson S, Carter SD: \u003cb\u003eHuman articular chondrocytes express three facilitative glucose transporter isoforms: GLUT1, GLUT3 and GLUT9\u003c/b\u003e. Cell Biol Int 2002, \u003cb\u003e26\u003c/b\u003e(3):297\u0026ndash;300.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVannucci SJ, Seaman LB, Vannucci RC: \u003cb\u003eEffects of hypoxia-ischemia on GLUT1 and GLUT3 glucose transporters in immature rat brain\u003c/b\u003e. J Cereb Blood Flow Metab 1996, \u003cb\u003e16\u003c/b\u003e(1):77\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eV\u0026aacute;zquez-Mosquera M, Rego-P\u0026eacute;rez I, Soto-Hermida A, Fern\u0026aacute;ndez-Moreno M, Fern\u0026aacute;ndez-Tajes J, Cort\u0026eacute;s-Pereira E, Rela\u0026ntilde;o-Fern\u0026aacute;ndez S, Oreiro-Villar N, Fern\u0026aacute;ndez-L\u0026oacute;pez C, Blanco FJO \u003cem\u003eet al\u003c/em\u003e: \u003cb\u003eNew insights into the molecular basis of the metabolic alterations in the osteoarthritis (OA) disease\u003c/b\u003e. 2013, \u003cb\u003e21\u003c/b\u003e:S170.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosa SC, Goncalves J, Judas F, Mobasheri A, Lopes C, Mendes AF: \u003cb\u003eImpaired glucose transporter-1 degradation and increased glucose transport and oxidative stress in response to high glucose in chondrocytes from osteoarthritic versus normal human cartilage\u003c/b\u003e. Arthritis Res Ther 2009, \u003cb\u003e11\u003c/b\u003e(3):R80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoldring MB: \u003cb\u003eUpdate on the biology of the chondrocyte and new approaches to treating cartilage diseases\u003c/b\u003e. Best Pract Res Clin Rheumatol 2006, \u003cb\u003e20\u003c/b\u003e(5):1003\u0026ndash;1025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng L, Zhang Z, Sheng P, Mobasheri A: \u003cb\u003eThe role of metabolism in chondrocyte dysfunction and the progression of osteoarthritis\u003c/b\u003e. Ageing Res Rev 2021, \u003cb\u003e66\u003c/b\u003e:101249.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKonttinen YT, Mandelin J, Li TF, Salo J, Lassus J, Liljestrom M, Hukkanen M, Takagi M, Virtanen I, Santavirta S: \u003cb\u003eAcidic cysteine endoproteinase cathepsin K in the degeneration of the superficial articular hyaline cartilage in osteoarthritis\u003c/b\u003e. Arthritis Rheum 2002, \u003cb\u003e46\u003c/b\u003e(4):953\u0026ndash;960.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRazaq S, Wilkins RJ, Urban JP: \u003cb\u003eThe effect of extracellular pH on matrix turnover by cells of the bovine nucleus pulposus\u003c/b\u003e. Eur Spine J 2003, \u003cb\u003e12\u003c/b\u003e(4):341\u0026ndash;349.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilkins RJ, Hall AC: \u003cb\u003eControl of matrix synthesis in isolated bovine chondrocytes by extracellular and intracellular pH\u003c/b\u003e. J Cell Physiol 1995, \u003cb\u003e164\u003c/b\u003e(3):474\u0026ndash;481.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHunter DJ, McDougall JJ, Keefe FJ: \u003cb\u003eThe symptoms of osteoarthritis and the genesis of pain\u003c/b\u003e. Rheum Dis Clin North Am 2008, \u003cb\u003e34\u003c/b\u003e(3):623\u0026ndash;643.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArra M, Swarnkar G, Ke K, Otero JE, Ying J, Duan X, Maruyama T, Rai MF, O\u0026rsquo;Keefe RJ, Mbalaviele G \u003cem\u003eet al\u003c/em\u003e: \u003cb\u003eLDHA-mediated ROS generation in chondrocytes is a potential therapeutic target for osteoarthritis\u003c/b\u003e. Nature Communications 2020, \u003cb\u003e11\u003c/b\u003e(1):3427.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKan S, Duan M, Liu Y, Wang C, Xie J: \u003cb\u003eRole of Mitochondria in Physiology of Chondrocytes and Diseases of Osteoarthritis and Rheumatoid Arthritis\u003c/b\u003e. Cartilage 2021, \u003cb\u003e13\u003c/b\u003e(2_suppl):1102S-1121S.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBonora M, Patergnani S, Rimessi A, De Marchi E, Suski JM, Bononi A, Giorgi C, Marchi S, Missiroli S, Poletti F \u003cem\u003eet al\u003c/em\u003e: \u003cb\u003eATP synthesis and storage\u003c/b\u003e. Purinergic Signal 2012, \u003cb\u003e8\u003c/b\u003e(3):343\u0026ndash;357.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQu J, Lu D, Guo H, Miao W, Wu G, Zhou M: \u003cb\u003ePFKFB3 modulates glycolytic metabolism and alleviates endoplasmic reticulum stress in human osteoarthritis cartilage\u003c/b\u003e. Clin Exp Pharmacol Physiol 2016, \u003cb\u003e43\u003c/b\u003e(3):312\u0026ndash;318.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmolle M, Prior AE, Brown AE, Cooper A, Byron O, Lindsay JG: \u003cb\u003eA new level of architectural complexity in the human pyruvate dehydrogenase complex\u003c/b\u003e. J Biol Chem 2006, \u003cb\u003e281\u003c/b\u003e(28):19772\u0026ndash;19780.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Shen X, Yan Y, Li H: \u003cb\u003ePyruvate dehydrogenase kinases (PDKs): an overview toward clinical applications\u003c/b\u003e. Biosci Rep 2021, \u003cb\u003e41\u003c/b\u003e(4).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcFate T, Mohyeldin A, Lu H, Thakar J, Henriques J, Halim ND, Wu H, Schell MJ, Tsang TM, Teahan O \u003cem\u003eet al\u003c/em\u003e: \u003cb\u003ePyruvate dehydrogenase complex activity controls metabolic and malignant phenotype in cancer cells\u003c/b\u003e. J Biol Chem 2008, \u003cb\u003e283\u003c/b\u003e(33):22700\u0026ndash;22708.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim JW, Tchernyshyov I, Semenza GL, Dang CV: \u003cb\u003eHIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia\u003c/b\u003e. Cell Metab 2006, \u003cb\u003e3\u003c/b\u003e(3):177\u0026ndash;185.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJeoung NH: \u003cb\u003ePyruvate Dehydrogenase Kinases: Therapeutic Targets for Diabetes and Cancers\u003c/b\u003e. Diabetes Metab J 2015, \u003cb\u003e39\u003c/b\u003e(3):188\u0026ndash;197.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYudoh K, Nakamura H, Masuko-Hongo K, Kato T, Nishioka K: \u003cb\u003eCatabolic stress induces expression of hypoxia-inducible factor (HIF)-1 alpha in articular chondrocytes: involvement of HIF-1 alpha in the pathogenesis of osteoarthritis\u003c/b\u003e. Arthritis Res Ther 2005, \u003cb\u003e7\u003c/b\u003e(4):R904-914.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoberts DJ, Miyamoto S: \u003cb\u003eHexokinase II integrates energy metabolism and cellular protection: Akting on mitochondria and TORCing to autophagy\u003c/b\u003e. Cell Death Differ 2015, \u003cb\u003e22\u003c/b\u003e(2):248\u0026ndash;257.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBustamante MF, Oliveira PG, Garcia-Carbonell R, Croft AP, Smith JM, Serrano RL, Sanchez-Lopez E, Liu X, Kisseleva T, Hay N \u003cem\u003eet al\u003c/em\u003e: \u003cb\u003eHexokinase 2 as a novel selective metabolic target for rheumatoid arthritis\u003c/b\u003e. Ann Rheum Dis 2018, \u003cb\u003e77\u003c/b\u003e(11):1636\u0026ndash;1643.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCiscato F, Ferrone L, Masgras I, Laquatra C, Rasola A: \u003cb\u003eHexokinase 2 in Cancer: A Prima Donna Playing Multiple Characters\u003c/b\u003e. Int J Mol Sci 2021, \u003cb\u003e22\u003c/b\u003e(9).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou KL, Zhu ZH, Zhou JP, Zhao JJ, Zhang Y, Jiang B: \u003cb\u003eIncreased hexokinase-2 as a novel biomarker for the diagnosis and correlating with disease severity in rheumatoid arthritis\u003c/b\u003e. Medicine (Baltimore) 2021, \u003cb\u003e100\u003c/b\u003e(25):e26504.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlaney Davidson EN, van der Kraan PM, van den Berg WB: \u003cb\u003eTGF-beta and osteoarthritis\u003c/b\u003e. Osteoarthritis Cartilage 2007, \u003cb\u003e15\u003c/b\u003e(6):597\u0026ndash;604.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen J, Li S, Chen D: \u003cb\u003eTGF-beta signaling and the development of osteoarthritis\u003c/b\u003e. Bone Res 2014, 2.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan der Kraan PM: \u003cb\u003eDifferential Role of Transforming Growth Factor-beta in an Osteoarthritic or a Healthy Joint\u003c/b\u003e. J Bone Metab 2018, \u003cb\u003e25\u003c/b\u003e(2):65\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang C, Silverman RM, Shen J, O'Keefe RJ: \u003cb\u003eDistinct metabolic programs induced by TGF-beta1 and BMP2 in human articular chondrocytes with osteoarthritis\u003c/b\u003e. J Orthop Translat 2018, \u003cb\u003e12\u003c/b\u003e:66\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZahra K, Dey T, Ashish, Mishra SP, Pandey U: \u003cb\u003ePyruvate Kinase M2 and Cancer: The Role of PKM2 in Promoting Tumorigenesis\u003c/b\u003e. Front Oncol 2020, \u003cb\u003e10\u003c/b\u003e:159.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cdiv class=\"gridtable\"\u003e\n\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cdiv class=\"SimplePara\"\u003ePrimary antibodies used in the immunohistochemical analyses in the present study\u003c/div\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003ePrimary Antibody\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eHost\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv class=\"SimplePara\"\u003eSupplier\u003c/div\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cdiv 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class=\"SimplePara\"\u003e1:500\u003c/div\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\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":"osteoarthritis, oxamate, glycolysis, chondrocytes, lactate dehydrogenase A","lastPublishedDoi":"10.21203/rs.3.rs-2540780/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2540780/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Osteoarthritis (OA) is the most common form of joint disorder and arthritis worldwide. Joint pain and dysfunction are associated with this multifactorial disease characterized by the gradual degeneration of articular cartilage. The metabolic reprogramming of osteoarthritic chondrocytes to glycolysis instead of oxidative phosphorylation results in reduced ATP and lactate accumulation. When the glycolytic metabolite pyruvate is converted into lactate by lactate dehydrogenase A (LDHA), cartilage degeneration occurs. In the present study, we examined the chondroprotective effects of the LDHA inhibitor, oxamate on experimental OA rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Anterior cruciate ligament transection (ACLT)-induced ACLT-rats received an intraarticular (IA) injection of oxamate once a week for 5 weeks from the 10\u003csup\u003eth\u003c/sup\u003e to 14\u003csup\u003eth\u003c/sup\u003e after surgery. Animals were divided into four groups as follows: Sham, ACLT, ACLT + oxamate (0.25mg/kg), and ACLT + oxamate (2.5mg/kg). The results showed that an IA injection of oxamate significantly reduced weight-bearing defects and knee swelling in ACLT-rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Histopathological analyses showed that oxamate caused significantly less cartilage degeneration than ACLT alone. Moreover, IA oxamate exerts hypertrophic effects in the chondrocyte of articular cartilage by inhibiting glucose-transporter 1, glucose-transporter 3, pyruvate kinase, LDHA, pyruvate dehydrogenase kinase 1, and pyruvate dehydrogenase kinase 2. Terminal deoxynucleotidyl transferase dUTP nick end labeling revealed that oxamate significantly reduced chondrocyte apoptosis in articular cartilage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eWe propose that oxamate is beneficial for patients with OA and is associated with regulating glycolysis-related protein expression. In future clinical applications, our findings will provide new insights of LHDA inhibitor, oxamate into delaying strategies for OA progression.\u003c/p\u003e","manuscriptTitle":"The effects of intra-articular oxamate on anterior cruciate ligament transection-induced experimental osteoarthritic rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-02-06 14:38:51","doi":"10.21203/rs.3.rs-2540780/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":"43b2fc10-3c21-46d1-83b6-5fb5cfebdcf3","owner":[],"postedDate":"February 6th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-03-27T20:44:27+00:00","versionOfRecord":[],"versionCreatedAt":"2023-02-06 14:38:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2540780","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2540780","identity":"rs-2540780","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}