Effect of needle knife on autophagy and matrix degradation of chondrocytes in rabbit knee osteoarthritis

To observe the effect of needle knife on chondrocyte autophagy and expressions of autophagy-related protein and mammalian target of rapamycin (mTOR) in rats with knee osteoarthritis (KOA), and to explore the possible mechanism of needle knife for KOA. A total of 42 SD rats were randomly divided into a normal group, a model group and a needle knife group, 14 rats in each group. Except for the normal group, the other two groups were injected with the mixture of papain and L-cysteine into the left hind knee joint to establish the KOA model. After modeling, the rats in the needle knife group were treated with needle knife at strip or nodule around the quadriceps femoris and medial and lateral collateral ligament on the affected side, once a week for 3 times (3 weeks). The changes of left knee circumference in each group were observed; the chondrocytes and ultrastructure of left knee joint were observed by HE staining and electron microscope; the mRNA and protein expressions of autophagy-related genes (Atg5, Atg12, Atg4a), Unc-51 like autophagy activated kinase 1 (ULK1), autophagy gene Beclin-1 and mTOR in left knee cartilage were detected by real-time fluorescence quantitative PCR and Western blot. After modeling, the left knee circumferences in the model group and the needle knife group were increased compared with those before modeling and in the normal group (P<0.05); after intervention, the left knee circumference in the needle knife group was smaller than that in the model group and after modeling (P<0.05). Compared with the normal group, the number of chondrocytes was decreased, and a few cells swelled, nuclei shrank, mitochondria swelled and autophagosomes decreased in the model group; compared with the model group, the number of chondrocytes was increased , and most cell structures returned to normal, and autophagosomes was increased. Compared with the normal group, the mRNA and protein expressions of Atg5, Atg12, Atg4a, Beclin-1 and ULK1 in the knee cartilage in the model group were decreased (P<0.05); compared with the model group, the expressions of the above indexes in the needle knife group were increased (P<0.05). Compared with the normal group, the mRNA and protein expressions of mTOR in the knee cartilage in the model group were increased (P<0.05); compared with the model group, the expressions of the above indexes in the needle knife group were decreased (P<0.05). The needle knife intervention could improve knee cartilage injury in rats with KOA, and its mechanism may be related to reducing the expression of mTOR and up-regulating the expressions of Atg5, Atg12, Atg4a, ULK1 and Beclin-1, so as to promote chondrocyte autophagy and delay the aging and degeneration of chondrocytes.

We have demonstrated that needle knife (acupotomy) treatment can improve knee osteoarthritis (KOA) in rabbits. The present study was designed to examine its effect on expression of phosphorylated focal adhesion kinase (p-FAK), phosphinositides 3 kinase (p-PI 3 K) and Aggrecan genes and proteins in the knee-joint cartilage tissues of KOA rabbits, so as to explore its partial molecular mechanism underlying improvement of KOA. Forty-nine New Zealand male rabbits were randomly divided into normal control, model, model +inhibitor, needle knife, needle knife+ inhibitor, electroacupuncture (EA), EA +inhibitor groups (n=7 in each). The KOA model was established by modified Videman method (left hindlimb extension immobilization and ankle dorsal flexion 60°). Acupotomy relaxing manipulation was applied to the lateral collateral ligament and patellar ligament of the left knee-joint, two times a week for 4 weeks, and EA (2 Hz/100 Hz, 3 mA) was applied to the left "Liangmen" (ST 21), "Xuehai" (SP 10), "Neixiyan " (EX-LE 4) and "Dubi" (ST 35) for 20 min, three times a week, for 4 weeks. About 2 h before every needle-knife or EA treatment or at the corresponding time-point, intra-articular cavity injection of PF-562271(a specific antagonist of FAK, 200 μmol/L, 0.5 mL)was performed in the three inhibitor groups. The expression levels of p-FAK, p-PI 3 K, Aggrecan genes and proteins in the cartilage tissues were measured with quantitative Real-time PCR and Western blot, separately. After modeling, the expression levels of p-FAK and p-PI 3 K genes and proteins were significantly up-regulated (P<0.05, P<0.01), while those of Aggrecan protein and mRNA considerably down-regulated in the model group in comparison with the normal group (P<0.01). Following 4 weeks' needle-knife or EA treatment, the expression levels of p-FAK and p-PI 3 K and Aggrecan proteins in both EA and needle-knife groups, and Aggrecan mRNA in the needle knife group were significantly up-regulated (P<0.05, P<0.01). After administration of p-FAK antagonist, modeling-induced upregulation of expression of p-FAK mRNA and protein and p-PI 3 K protein, as well as modeling-induced down-regulation of Aggrecan mRNA and protein were significantly suppressed in the model+inhibitor group (P<0.05, P<0.01), and needle knife-induced and EA-induced up-regulation of expression of p-FAK, p-PI 3 K and Aggrecan mRNAs and proteins was notably suppressed respectively in comparison with the needle knife and EA groups (P<0.05, P<0.01). The effect of needle knife was significantly superior to that of EA in up-regulating p-PI 3 K and Aggrecan mRNAs as well as p-FAK, p-PI 3 K and Aggrecan proteins (P<0.05, P<0.01). Needle knife intervention can up-regulate the expression levels of p-FAK, p-PI 3 K and Aggrecan proteins and mRNAs in the cartilage tissue of the knee-joint in KOA rabbits, suggesting an involvement of FAK-PI 3 K signaling in the needle knife-induced improvement of KOA.

To explore the effect of acupotomy intervention on autophagy of chondrocytes in rabbits with knee osteoarthritis (KOA), and to determine the possible mechanisms of acupotomy to alleviate cartilage degeneration. The modified Videman method was used to construct a KOA rabbit model. After modeling, 40 rabbits were randomly divided into 4 groups by a random number table: control; KOA (model); KOA + acupotomy (acupotomy), and KOA + sham acupotomy (sham), 10 in each group. After a 3-week treatment course, the knee joint activity was determined by the modified Lequesne MG index. Hematoxylin-eosin staining staining was used to examine the morphological changes of chondrocytes. Autophagy of chondrocytes was observed by transmission electron microscopy. The surface morphology of cartilage tissue was observed by scanning electron microscope. The mRNA and protein levels of AMP kinase/mammalian target of rapamycin/Unc-51 (AMPK/mTOR/ULK1) signal pathway key proteins, autophagy-related factor Beclin-1 and microtubule-associated protein 1A/1B light chain 3 (LC3) in rabbit knee cartilage were assessed by real-time fluorescence quantitative polymerase chain reaction and Western blot, respectively. The modified Lequesne MG score of acupotomy group was significantly lower than that of model group (P<0.05). Pathological results showed that chondrocyte autophagy decreased and cartilage surface was rough in the model group, which recovered after acupotomy treatment. The mRNA expressions of AMPK, ULK1, Beclin-1 and the protein levels of p-AMPK, p-ULK1, Beclin-1, and LC3 II/LC3 I were decreased in the model group, while the mRNA and protein expressions of mTOR were increased (P<0.01). However, acupotomy treatment reversed these abnormal changes (P<0.05). Acupotomy could effectively up-regulate the expressions of AMPK, ULK1 and Beclin1, reduce the expression of mTOR, promote autophagy, and alleviate joint degeneration. Acupotomy is a promising complementary and alternative therapy for KOA.

To observe the effects of acupotomy intervention on the behavior, morphology and tensile mechanics of knee osteoarthritis (KOA) rabbits, and to explore the biomechanical effects of acupotomy on KOA. Twenty-four New Zealand male rabbits were randomly divided into four groups: normal group, model group, electroacupuncture group and needle-knife group, with 6 rabbits in each group. In each model group, rabbit KOA model was established by fixing Videman's left hind limb in straight position for 6 weeks. In the electroacupuncture group, rats were treated left on Liang Qiu, Xue Hai, Nei Xi Yan and Wai Xi Yan 3 times a week for 3 weeks. In the acupotomology group, the left quadriceps femoris tendon was released with acupotomology, and the treatment was once a week for 3 weeks. Behavioral tests were performed using Lequesne MG knee joint evaluation method one week after the end of modeling and one week after the end of treatment, and HE staining and mechanical tests were performed one week after the end of treatment. Behavioral observation before treatment showed that there were significant differences in local pain, gait response, joint activity and joint swelling between the normal group and the model group(P<0.05), while there was no significant difference among the model group, electro-acupuncture group and needle-knife group(P>0.05). After treatment, the results showed that there were significant differences in local pain, gait response, joint activity and joint swelling among model group, electro-acupuncture group and needle-knife group compared with normal group(P<0.05); In local pain, the electro-acupuncture group was lower than the model group, and there was no significant difference(P>0.05); there was significant difference between needle knife group and model group(P<0.05); there was no significant difference between electro-acupuncture group and needle-knife group(P>0.05). In gait change, there was significant difference between model group and electro-acupuncture group(P<0.05); there was no significant difference between needle-knife group and model group(P>0.05). In joint activity, there was significant difference between electro-acupuncture group and model group(P<0.05). In joint swelling, compared with model group, there was significant difference on electro-acupuncture group and electro-knife group(P<0.01), but there was no significant difference between the electro-acupuncture group and the needle-knife group(P>0.05). Mechanics: Compared with the blank group, the ultimate load of the model group decreased significantly(P<0.01), the ultimate load of the electro-acupuncture group decreased(P>0.05), and the ultimate load of the needle-knife group increased(P>0.05). Compared with the model group, the ultimate load of the electro-acupuncture group increased significantly(P<0.05), and the ultimate load of the needle-knife group increased significantly (P<0.01). Compared with the electro-acupuncture group, the ultimate load of the needle-knife group increased(P>0.05). Compared with the blank group, the maximum displacement of the model group decreased significantly(P<0.01), and the maximum displacement of the electro-acupuncture group and the needle-knife group decreased(P>0.05). Compared with the model group, the maximum displacement of the electro-acupuncture group increased(P>0.05), and the maximum displacement of the needle-knife group increased significantly(P<0.05). Compared with the electro-acupuncture group, the maximum displacement of the needle-knife group increased(P>0.05). There was no significant difference in stiffness among groups(P>0.05). Acupotomy intervention can significantly change the behavior and morphology, significantly improve the mechanical properties of quadriceps femoris tendon stretch, and exert its biomechanical effects to achieve the purpose of treating KOA.

To observe the effect of needle-knife on the chondrocyte apoptosis of knee joint in rabbits with knee osteoarthritis (KOA) based on the CircSERPINE2-miR-1271-5P-E26 specific transformation-related gene (ERG) axis, and to explore the mechanism of needle-knife for KOA. Thirty-six New Zealand white rabbits were randomly divided into a normal group, a model group, a needle-knife group and a sham needle-knife group, 9 rabbits in each group. The rabbits in the model group, the needle-knife group and the sham needle-knife group were treated with modified Videman method to prepare KOA model. After successful modeling, the rabbits in the needle-knife group were treated with needle-knife at cord adhesion and nodules near quadriceps femoris tendon and internal and external collateral ligament on the affected knee joint; the rabbits in the sham needle-knife group were treated with sham needle-knife baside the needle insertion point of the needle-knife group (needle-knife was only inserted, without any operation). The treatment was given once a week, 3 times in total. The Lequesne MG behavioral score was used to evaluate the knee joint damage in each group before and after intervention. After intervention, HE staining and transmission electron microscopy were used to observe the cartilage tissue morphology and ultrastructure of chondrocytes in the knee joint in each group; TUNEL method was used to detect the level of chondrocyte apoptosis in the knee joint; real-time fluorescence quantitative PCR was used to detect the expression of CircSERPINE2, miR-1271-5P and ERG mRNA in knee cartilage tissue in each group. After intervention, compared with the normal group, the Lequesne MG behavioral score in the model group was increased (P<0.01). Compared with the model group and the sham needle-knife group, the Lequesne MG behavioral score in the needle-knife group was decreased (P<0.01). In the model group and the sham needle-knife group, the number of chondrocytes and organelles was decreased, the cell nucleus was shrunk, mitochondria was swelling or disappeared; in the needle-knife group, the number of chondrocytes and organelles was increased, the cell nucleus was not obviously shrunk and the mitochondria was not obviously swelling. Compared with the normal group, the level of chondrocyte apoptosis in the model group was increased (P<0.01); compared with the model group and the sham needle-knife group, the level of chondrocyte apoptosis in the needle-knife group was decreased (P<0.01, P<0.05). Compared with the normal group, the expression of CircSERPINE2 and ERG mRNA in the model group was decreased (P<0.01), and the expression of miR-1271-5P mRNA was increased (P<0.01); compared with the model group and the sham needle-knife group, the expression of CircSERPINE2 and ERG mRNA in the needle-knife group was increased (P<0.01), and the expression of miR-1271-5P mRNA was decreased (P<0.01). Needle-knife could reduce the knee joint damage and chondrocyte apoptosis in KOA rabbits, which may be related to up-regulating the expression of CircSERPINE2 and ERG mRNA, and inhibiting the expression of miR-1271-5P mRNA.

To observe the effect of warm acupuncture on the expression of Janus protein tyrosine kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway and inflammatory factors of articular cartilage in rabbits with knee osteoarthritis (KOA), so as to explore its underlying mechanisms in improving KOA. New Zea-land rabbits were randomly divided into blank, model, warm acupuncture and medication groups (12 rabbits in each group). The KOA model was prepared by using the right hind limb tubular plaster extension fixation method. The rabbits in the warm acupuncture group received acupuncture of "Neixiyan"(EX-LE4),"Waixiyan"(ST35),"Heding"(EX-LE2) and "Zusanli"(ST36), followed by attaching an ignited moxa-stick segment to the acupuncture-handle. The treatment was conducted for 15 min, once a week for 4 weeks. The rabbits in the medication group received gavage of diclofenac sodium solution(0.35 mg/kg), once daily for 4 weeks. The dysfunction severity state of the rabbit's knee-joint was evaluated using Lequesne scale (0-3 points), and the histopathological changes of cartilage were observed under microscope after H.E. staining and the state of distribution of chondrocytes in different layers and the extracellular matrix was assessed using Mankin score (0-6 points). The contents of serum interleukin (IL)-6, tumor necrosis factor-α (TNF-α) and matrix metalloproteinase-9 (MMP-9) were measured by using ELISA, and the expression levels of p-JAK2/JAK2, p-STAT3/STAT3 and MMP-9 in knee cartilage tissue were detected using Western blot. Compared with the blank group, the Lequesne score, Mankin score, and the contents of serum IL-6, TNF-α and MMP-9, and the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, and the expression level of MMP-9 protein in knee cartilage tissue were significantly increased in the model group (P<0.01). In comparison with the model group, the Lequesne score, Mankin score, contents of serum IL-6, TNF-α and MMP-9, and the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, and the expression of MMP-9 protein in knee cartilage tissue were notably decreased in both the warm acupuncture and medication groups (P<0.01,P<0.05). The levels of Lequesne score, Mankin score, contents of serum IL-6, TNF-α and MMP-9, and the ratios of p-JAK2/JAK2 and p-STAT3/STAT3 in knee cartilage tissue were significantly lower in the warm acupuncture group than in the medication group (P<0.01, P<0.05). No significant difference was found between the warm acupuncture and medication groups in the expression of MMP-9 protein (P>0.05). Outcomes of H.E. showed injury of the perichondrium of knee joint, obvious reduction of the cartilage matrix staining, cystic changes, clustered and disordered arrangement and severe pyknosis and necrosis of the surface cells with reduction of number of cells and increase of vacuoles in the model group, which was milder in both warm acupuncture and medication groups. Warm acupuncture can improve motor function and reduce cartilage injury in KOA rabbits, which may be related to its functions in inhibiting the secretion of pro-inflammatory factors and regulating JAK2/STAT3 signaling and downregulating MMP-9 expression in the cartilage tissue.

Objective: To observe the effect of autophagy of condylar chondrocytes on apoptosis in temporomandibular joint osteoarthritis (TMJOA) of rats. Methods: Fourty male 2-month-old SPF SD rats were equally divided into sham group (n=20) and experimental group (n=20). UAC metal prosthesis was cemented to the left incisors of maxilla and mandible of the rats in experimental group rats. After 8 weeks, all rats were sacrificed and the temporomandibular joint was taken. Two groups of rat condylar chondrocytes were extracted and cultured in vitro to the third generation. Immunofluorescence technique was used to detect the levels of collagen Ⅱ and matrix metalloproteinase-13 (MMP-13) in chondrocytes. The level of light chain-3 (LC-3), an autophagy marker of chondrocytes, was detected. Immunohistochemical technique was used to detect the level glycogenin-1, a glycogen formation marker of chondrocyte, was detected. The level of caspase-3, an apoptosis marker of chondrocyte, was also detected. Tunel technique was used to detect the apoptosis rate of the two groups at 72 h. Cracking cell extraction of total protein, Western-blotting (WB) technology to detect the levels of collagen Ⅱ, MMP -13, LC-3, glycogenin-1, caspase-3 and make gray analysis. Results: Compared with sham group, the level of collagen Ⅱ decreased, MMP-13 increased, LC-3 decreased, glycogenin-1 increased and caspase-3 increased in experimental group. The apoptosis rate of chondrocytes in experimentaal group [ (17.3±4.4) %] at 72h was higher than that in control group [ (5.6±2.1) %](t=10.732, P<0.001) .WB bands gray statistical results show that the level of collagen Ⅱ in chondrocytes of experimental group (0.43±0.21) was lower than that of control group (0.71±0.26) (t=2.409, P=0.043) , the level of MMP-13 in chondrocytes of experimental group (0.73±0.31) was higher than that of control group (0.24±0.10) (t=3.364, P=0.010) , the level of LC-3 in chondrocytes of experimental group (0.09±0.04) was lower than that of control group (0.39±0.18) (t=3.638, P=0.007) , the level of glycogenin-1 in chondrocytes of experimental group (0.68±0.30) was higher than that of control group (0.29±0.17) (t=2.529, P=0.035) , the level of caspase-3 in chondrocytes of experimental group (0.19±0.08) was higher than that of control group (0.05±0.02) (t=3.796, P=0.005) . Conclusions: The level of autophagy of condylar chondrocytes in temporomandibular joint of rats decreased, glycogen accumulation increased, the rate of chondrocyte apoptosis increased, and the number of chondrocytes decreased, resulting in degeneration of condylar cartilage tissue.

To observe the application value of MR T2 mapping for evaluating the effect of warm acupuncture-moxibustion on articular cartilage degeneration, and to observe the relationship between T2 value and expression of matrix metalloproteinases (MMP)-1 and MMP-13 of chondrocytes in rabbits with early knee osteoarthritis (KOA). Thirty male New Zealand rabbits were randomly divided into blank control, KOA model and warm acupuncture-moxibustion groups, with 10 rabbits in each group. The early KOA model was established by right hind limb tubular plaster extension fixation method for 2 weeks. The rabbits of the warm acupuncture-moxibustion group received warm acupuncture-moxibustion stimulation at "Heding"(EX-LE2), "Neixiyan"(EX-LE4), "Waixiyan" (EX-LE5) and"Zusanli"(ST36) on the right hind limb for 15 min, once a day for 2 weeks. After intervention, MR T2 mapping of the right knee joint was performed in each group. The H.E. staining was used to evaluate the histopathological changes of cartilage, followed by giving a score according to the standards of Mankin scoring. The TUNEL method was used to analyze the apoptosis state of chondrocytes, and the positive expressions of MMP-1 and MMP-13 in the articular cartilage were detected by immunohistochemical staining. Compared with the blank control group, the Mankin score, chondrocyte apoptosis index, T2 value and the positive expressions of MMP-1 and MMP-13 in the cartilage tissue were significantly increased in the model group (P<0.01). Compared with the model group, the Mankin score, chondrocyte apoptosis index, T2 value and the positive expressions of MMP-1 and MMP-13 in the cartilage tissue were markedly decreased in the warm acupuncture-moxibustion group (P<0.01). The T2 value was positively correlated with the expression levels of MMP-1 and MMP-13 (P<0.01). H.E. staining showed disordered arrangement of chondrocytes and thinner cartilage layer in the model group, and a clear and relative ordered arrangement of chondrocyte in the warm acupuncture-moxibustion group. Warm acupuncture-moxibustion can reduce the T2 value of articular cartilage in early KOA rabbits, which is positively correlated with the decreased expression of MMP-1 and MMP-13 in the extracellular matrix of cartilage. The MR T2 mapping has certain value in evaluating the effect of warm acupuncture-moxibustion on KOA rabbits with early cartilage degeneration.

This study aims to investigate the effects of Jiawei Duhuo Jisheng Mixture on mitochondrial autophagy in the cartilage of rabbits with knee osteoarthritis(KOA) based on the PTEN-induced kinase 1(PINK1)/Parkinson protein(Parkin) pathway and explore its potential mechanism in improving cartilage lesions. A KOA model was established by fixing a high-molecular resin plaster bandage on the right hind limb of the rabbits for six weeks. After successful modeling, the modeling group was randomly divided into a model group, a celecoxib group, and low-and high-dose groups of Jiawei Duhuo Jisheng Mixture, with eight rabbits in each group. The celecoxib group was administered celecoxib by gavage at a single dose of 0.009 3 g·kg~(-1). The low-and high-dose groups of Jiawei Duhuo Jisheng Mixture were given Jiawei Duhuo Jisheng Mixture at single doses of 6.8 mL·kg~(-1)(4.515 2 g·kg~(-1)) and 27.2 mL·kg~(-1)(18.060 8 g·kg~(-1)), respectively. Administered once daily for six weeks, the rabbits in each group then underwent behavioral testing. After sample collection, the gross morphological changes of the knee articular cartilage were observed with the naked eye. Hematoxylin-eosin(HE) staining was used to detect pathological changes in cartilage tissue, which were quantitatively evaluated by using the Lequesne MG score, Pelletier score, and Mankin score. Transmission electron microscopy was used to observe the ultrastructural changes of chondrocyte mitochondria. Flow cytometry was used to detect the mitochondrial membrane potential(Δψm) and the average fluorescence intensity of reactive oxygen species(ROS) in chondrocytes and calculate the percentage of cells with low Δψm. Western blot was used to detect the expression level of mitochondrial autophagy-related proteins in cartilage tissue, including PINK1, Parkin, selective autophagy adapter protein 62(P62), light chain 3(LC3)Ⅱ/LCⅠ, mitochondrial outer membrane translocase 20(TOM20), collagen type Ⅱ alpha 1(COL2A1), aggrecan(ACAN), matrix metalloproteinase(MMP)-9, and MMP-13. Immunohistochemistry(IHC) was used to detect the expression of PINK1, Parkin, and LC3B in cartilage tissue. The results showed that, compared with the blank group, the model group exhibited marked knee joint swelling and damage, tissue fibrosis, sparse chondrocyte distribution, and indistinct and incomplete tide marks. The Lequesne MG, Pelletier, and Mankin scores increased significantly. Autophagosomes were reduced, and mitochondria were morphologically abnormal. The percentage of chondrocytes with low Δψm, ROS average fluorescence intensity, and the expression of P62, TOM20, MMP-9, and MMP-13 proteins in cartilage tissue rose significantly, while the expression of PINK1, Parkin, LC3Ⅱ/LCⅠ, COL2A1, and ACAN proteins in cartilage tissue decreased significantly. Compared with the model group, the celecoxib and both Jiawei Duhuo Jisheng Mixture groups showed improved knee articular cartilage surface, relatively intact tide marks, and denser chondrocytes. Their Lequesne MG, Pelletier, and Mankin scores dropped significantly. Autophagosomes increased, and mitochondrial swelling and damage eased. The percentage of chondrocytes with low Δψm, ROS average fluorescence intensity, and the expression of P62, TOM20, MMP-9, and MMP-13 proteins in cartilage tissue decreased significantly, while the expression of PINK1, Parkin, LC3Ⅱ/LCⅠ, COL2A1, and ACAN proteins in cartilage tissue increased significantly. In conclusion, Jiawei Duhuo Jisheng Mixture can effectively activate the PINK1/Parkin pathway to promote mitochondrial autophagy and alleviate articular cartilage damage in rabbits with KOA, thus slowing the progression of KOA.

To observe the protective effect of electroacupuncture (EA) on the knee joint cartilage tissue of rats with knee osteoarthritis (KOA), so as to explore the potential mechanism of miR-335-5p involved in the treatment of EA for KOA. A total of 42 adult male Sprague-Dawley (SD) rats were randomly divided into blank, model and EA groups, with 14 rats in each group. The model was established by injecting a sodium glutamate iodoacetate solution (80 μg/μL) into the right joint cavity. On the 15th day after the model establishment, rats in the EA group received EA intervention at "Yanglingquan" (GB34) and "Xuehai" (SP10) on the right side, and the needles were retained for 15 minutes. The intervention was carried out every other day for a total of two weeks. After the two-week intervention, the paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) of the rats were detected;a gait analyzer was used to detect the gait adaptability changes induced by pain;the pathological changes of the knee joint cartilage tissue were observed after hematoxylin-eosin (HE) staining;qPCR and Western blot were used to detect the expression levels of miR-335-5p, Dickkopf related protein 1 (DKK-1), β-catenin, and matrix metalloproteinase 13 (MMP-13) mRNAs and proteins in the cartilage tissue respectively;immunofluorescence staining was used to observe the positive fluorescence expression of β-catenin in the right knee joint cartilage tissue. In the model group, HE staining showed a large area of chondrocyte necrosis, with the dissolution and disappearance of cell nuclei, accompanied by a macrophages-predominant inflammatory cell infiltration;cartilage surface defects in local areas, necrosis of trabecular bone and proliferation of fibrous tissue in local areas, and new capillary formation in the proliferative areas were also observed. In the EA group, pathological manifestations such as inflammatory cell infiltration and fibrous tissue proliferation were significantly alleviated. Compared with the blank group, the PWMT, PWTL, the support duration, footprint area, and average ground pressure of the right hind paw, and the mRNA and protein expressions of DKK-1 in the right knee joint cartilage tissue were decreased (P<0.001) in the model group, while the expressions of miR-335-5p, mRNA and protein expressions of β-catenin and MMP-13, and the fluorescence intensity of β-catenin in the right knee joint cartilage tissue were increased (P<0.001, P<0.01). All the above indicators in the EA group were all reversed (P<0.001, P<0.05, P<0.01) in comparison with the model group. EA can significantly improve the pain symptoms of KOA rats, promote the recovery of motor function, and improve the pathological morphology of the knee joint cartilage tissue. This may be related to regulating the expression of miR-355-5p and Wnt/β-catenin pathway, as well as inhibiting the release of cartilage extracellular matrix-related degrading enzymes.

Needle knife therapy, a form of acupuncture and moxibustion, has been widely used in the clinical treatment of knee osteoarthritis (KOA). However, the mechanism is not clear. Therefore, we studied the mechanisms of action of needle knife intervention on KOA in rabbits, with the PERK-eIF2α-CHOP pathway as a starting point, in order to determine the mechanism underlying knee joint chondrocyte apoptosis. Apoptosis and ultrastructural changes in the articular cartilage were examined by pathological study and transmission electron microscopy, and PERK, eIF2α, and CHOP mRNA and protein levels were detected by qRT-PCR and western blot, respectively. PERK, eIF2α, and CHOP protein levels were significantly higher in the model group than in the normal group (P < 0.01) and were considerably downregulated in the needle knife and the medicine groups compared to the model group (P < 0.01). The eIF2α, p-eIF2α, and CHOP protein levels were not significantly different between the needle knife and medicine groups. The PERK, eIF2α, and CHOP mRNA levels in the drug group were higher than those in the needle knife group (P < 0.01). Needle knife therapy can regulate PERK-eIF2α-CHOP signaling pathway, which could be one of the mechanisms by which it affects chondrocyte apoptosis in KOA rabbits.

This study aimed to investigate the underlying mechanisms of Duhuo Jisheng Decoction(DJD) in the prevention and treatment of knee osteoarthritis(KOA). Forty SPF New Zealand rabbits were randomly divided using SPSS 26.0 software into five groups: blank group, model group, low-dose DJD group, high-dose DJD group, and high-dose DJD+phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathway activator group(high-dose DJD+740Y-P group), with eight rabbits in each group. Except for the blank group, the KOA model was established in the other groups using papain injection into the knee joint cavity combined with forced flexion of the knee joint. The day after modeling, the blank group and model group were given normal saline at 10 mL·kg~(-1) by gavage, the low-dose DJD group received DJD at 8.8 g·kg~(-1) by gavage, the high-dose DJD group received DJD at 35.2 g·kg~(-1) by gavage, and the high-dose DJD+740Y-P group received DJD at 35.2 g·kg~(-1) by gavage along with 740Y-P at 0.15 μmoL·kg~(-1) injected via the auricular vein. All groups received treatment continuously for four weeks. After modeling and intervention, behavioral observations were performed for all groups, and after the intervention, imaging assessments of the knee joints were conducted. Cartilage from the knee joints was collected, and gross morphological changes were observed. Pathological changes in cartilage tissue were examined using hematoxylin-eosin(HE) staining. The results of these observations were quantitatively evaluated using the Lequesne MG score, Kellgren-Lawrence(K-L) grading, Pelletier score, and Mankin score. ELISA was used to measure the levels of interleukin-1β(IL-1β), interleukin-18(IL-18), and matrix metalloproteinase 13(MMP13) in cartilage tissue. Real-time RT-PCR was used to detect the mRNA expression levels of PI3K, Akt, mTOR, Nod-like receptor protein 3(NLRP3), cysteine protease 1(caspase-1), and gasdermin D(GSDMD) in cartilage tissue. Western blot was employed to measure the protein expression levels of PI3K, Akt, mTOR, NLRP3, caspase-1, and GSDMD. The results showed that compared with the blank group, the model group exhibited significant knee joint degeneration, increased Lequesne MG score, K-L grading, Pelletier score, and Mankin score, elevated levels of IL-1β, IL-18, and MMP13 in cartilage tissue, activation of PI3K, Akt, and mTOR phosphorylation along with increased mRNA expression levels, and elevated protein and mRNA expression levels of NLRP3, caspase-1, and GSDMD. Compared with the model group, these indicators were reversed in both the low-dose and high-dose DJD groups, with the high-dose group showing greater decline degree than the low-dose DJD group. However, compared with the high-dose DJD group, the improvements in knee joint degeneration were less pronounced in the high-dose DJD+740Y-P group, with increased Lequesne MG score, K-L grading, Pelletier score, Mankin score, elevated levels of IL-1β, IL-18, and MMP13, activation of PI3K, Akt, and mTOR phosphorylation along with increased mRNA expression, and increased protein and mRNA expression levels of NLRP3, caspase-1, and GSDMD. In conclusion, DJD is effective and safe in the treatment of KOA, and its mechanism may be related to the inhibition of PI3K/Akt/mTOR signaling pathway-mediated pyroptosis in cartilage tissue, thereby improving knee joint bone structure, reducing the inflammatory response, and preventing cartilage matrix degradation.

To observe the effect of acupotomy on the piezoelectric mechanical gating ion channel component 1(Piezo1)/glutathione peroxidase 4(GPX4)/long-chain acyl coenzyme A synthetase 4(ACSL4)signaling axis of knee chondrocytes in knee osteoarthritis(KOA)rabbits, so as to explore its mechanisms underly improvement of KOA. Twenty-four male New Zealand rabbits were randomly divided into blank control, KOA model and acupotomy groups, with 8 rabbits in each group. The KOA model was established by immobilizing the left hind limb (with a plaster cast) in an extended position for 6 weeks according to the modified Videman method. For rabbits of the acupotomy group, the needle-knife release technique was applied to the musculotendon lesion points ("Hedingci" "Binwaishang" "Binneishang" "Chengfeijian" "Weiyangci" and "Yinlingci") of the left knee, once a week for 4 weeks. The degree of the left knee joint dysfunction was assessed by referring to Lequesne MG's functional disability scale (pain reaction [0-3 points], gait [0-3 points], joint motion [0-3 points] and swelling [0-2 points]) before and after the intervention. Histopathological changes of the left knee joint cartilage tissue (chondrocytes) were observed after H.E. staining. The mitochondrial structure of the cartilage chondrocytes was observed using transmission electron microscopy (TEM). The mRNA expression levels of Piezo1, GPX4 and ACSL4 in the cartilage tissue were detected by using real-time qPCR. The protein expression levels of Piezo1, GPX4 and ACSL4 in the cartilage tissue were detected by using Western blot. Compared with the blank control group, the model group showed a significant increase in the Lequesne MG score (P<0.01), expression levels of Piezo1 and ACSL4 mRNAs and proteins(P<0.01), and a notable decrease in the expression levels of GPX4 mRNA and protein(P<0.01, P<0.05).Compared with the model group, the acupotomy group had an evident decrease in the Lequesne MG score(P<0.05), expression levels of Piezo1 and ACSL4 mRNAs and proteins (P<0.01, P<0.05), and an evident increase in the expression levels of GPX4 mRNA and protein(P<0.01, P<0.05). The results of H.E. staining showed a rough surface of the cartilage of the left knee joint, wrinkled and broken nucleus, being sparse in number, scattered arrangement and with uneven level in the model group. Whereas in the acupotomy group, the cartilage surface of the knee joint was relatively flat, the nucleus was clear and increased in the number and the arrangement was in order, and the level was relatively distinct. The results of TEM showed shrunk and irregular chondrocytes, ruptured cell membrane, multiple dissolved cytoplasm, formation of some vacuoles, round or oval mitochondria with reduction in the number, shortening in the ridge, even disappearance, and dissolved matrix in a large area in the model group. In the acupotomy group, the chondrocytes were relatively regular in shape, with complete membrane and uniform cytoplasm, and the mitochondria were more in the number, with more uniform matrix and more ridges. Acupotomy can improve the motor function in KOA rabbits, which may be associated with its functions in inhibiting chondrocyte ferroptosis by regulating Piezo1/GPX4/ACSL4 signaling axis, and relieving cartilage injury.

This study aimed to assess the efficacy of needle-knife (NK) combined with etanercept (NKCE) in attenuating pain, inflammation, disease activity, and improving hip joint function in ankylosing spondylitis (AS) patients with hip joint involvement.Totally, 90 patients with active AS involving unilateral hip joint were enrolled and randomly assigned in 1:1:1 ratio to receive NKCE, NK or conventional drugs (control). The ESR, CRP, hip joint pain Visual Analogue Scale (VAS) score, bath ankylosing spondylitis disease activity index (BASDAI), bath ankylosing spondylitis functional index (BASFI), modified Harris hip score (mHHS), and range of motion (ROM) of affected hip joint were assessed at baseline (W0), after 1-week treatment (W1) and after 24-week treatment (W24).ESR and CRP were decreased in NKCE group compared with NK and control groups, while was not attenuated in NK group compared with control group. Regrading pain and disease activity, NKCE group presented a reduction in hip pain VAS score and BASDAI compared with NK and control groups, and NK group showed a decrease in hip pain VAS score and BASDAI compared with control group. Besides, BASFI was lowered in NKCE and NK groups compared with control group, but similar between NKCE and NK groups. mHHS and hip ROM were raised in NKCE and NK groups compared with control group, but similar between NKCE and NK groups.NKCE decreases hip pain, inflammation, disease activity and improves hip joint function in AS patients with hip joint involvement.

To observe the effect of electroacupuncture (EA) on knee function and symptoms, intestinal flora and TLR4/MyD88/NF-κB signaling pathway of articular cartilage in rabbits with knee osteoarthritis (KOA), so as to explore its potential mechanism underlying improvement of knee function by regulating the gut-joint axis response of KOA. Thirty-two Japanese white rabbits were randomly divided into blank, model, glucosamine and EA groups, with 8 rabbits in each group. The KOA model was reproduced by using modified Hulth method. The rabbits in the EA group were given EA (2 Hz/100 Hz, 0.5-1.5 mA) at bilateral "Xuehai"(SP10), "Yinlingquan"(SP9) and "Dubi"(ST35) for 15 min, once daily, 5 times a week for 4 weeks. The rabbits of the glucosamine group received glucosamine solution once daily, 5 times a week for 4 weeks. The knee function was evaluated by Lequesne MG index of severity for osteoarthritis (total scores of pain, swelling, range of motion, and gait) after modeling and treatment. H.E. staining was used to observe the histopathological changes of the knee cartilage tissue. Immunofluorescence histochemical method was used to detect the expressions of MyD88 and NF-κB p65, and Wes automated protein blot quantitative analysis technique was employed to detect the protein expressions of TLR4, MyD88 and NF-κB p65 in the knee cartilage tissue. Analysis of the species structure changes of intestinal flora was detected by 16S rDNA high-throughput sequencing. After modeling, compared with the blank group, the Lequesne MG score, immunofluorescence intensity of MyD88 and NF-κB p65, and the protein expression levels of TLR4, MyD88 and NF-κB p65 were significantly increased in the model group (P<0.05). H.E. staining showed thin and rough surface of the knee cartilage, clusters and disordered arrangement of chondrocytes, a few cellular nuclei being dark blue-purple in staining, with some nuclei being shrank, ruptured, and disappeared in the model group. In comparison with the model group, the Lequesne MG score, fluorescence intensity of MyD88 and NF-κB p65, and the protein expression levels of TLR4, MyD88 and NF-κB p65 were significantly decreased in both EA and glucosamine groups (P<0.05). The effects of EA were significantly superior to glucosamine in down-regulating the protein expression levels of TLR4, MyD88 and NF-κB (P<0.05). H.E. staining showed relatively complete surface of the knee cartilage, slightly hypertrophic chondrocytes with relatively clear nucleus and relatively complete tide line in both EA and glucosamine groups. Results of 16S rDNA sequencing showed that compared with the blank group, the Shannon index was obviously increased (P<0.05), the relative abundance of Proteobacteria, Actinobacteria, Escherichia coli-Shigella and Clostridium UCG-014 was elevated, and the relative abundance of Firmicutes, Verrucomicrobia, Comamonas and Acmea bacteria was decreased in the model group. In comparison with the model group, the Chao1 index and Shannon index were increased (P<0.05), the relative abundance of Proteobacteria, Actinobacteria and Escherichia coli-Shigella was decreased, while the relative abundance of Firmicutes, Comamonas and Nobilus was significantly increased in both EA and glucosamine groups. EA can significantly improve the symptoms and function of knee joint in KOA rabbits, which may be associated with its functions in inhibiting cartilage injury and inflammation reaction by regulating TLR4/MyD88/NF-κB signaling pathway and intestinal flora diversity through the "gut-joint" axis.