[Effect of acupotomy on mitophagy mediated by PINK1/Parkin pathway in cartilage of rabbits with knee osteoarthritis].

OBJECTIVE: To observe the effect of acupotomy on mitophagy mediated by PINK1/Parkin pathway in cartilage of rabbits with knee osteoarthritis (KOA), so as to explore its mechanism in inhibiting cartilage damage. METHODS: Twenty-one New Zealand rabbits were randomly divided into normal, model, and acupotomy groups, with 7 rabbits in each group. The KOA rabbit model was established by using the Videman method. Rabbits in the acupotomy group received regular acupotomy treatment around the knee joint nodules or tendons once a week for 3 consecutive weeks. HE staining and transmission electron microscopy were used to observe the morphological and ultrastructural changes in knee joint cartilage of rabbits. Flow cytometry was used to measure the mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) average fluorescence intensity in chondrocytes. Immunofluorescence was performed to detect the fluorescence intensity of LC3B, PINK1 and Parkin in cartilage tissue. Western blot was conducted to measure the protein expression levels of p62, LC3Ⅱ/Ⅰ, PINK1, and Parkin in cartilage tissue. RESULTS: Compared to the normal group, the model group showed fissures and tissue fibrosis on the surface of rabbit knee joint cartilages, loose distribution of chondrocytes, decreased autophagosomes, and abnormal mitochondrial morphology. The fluorescence intensity of LC3B, PINK1 and Parkin, the expression levels of LC3Ⅱ/Ⅰ, PINK1 and Parkin proteins in cartilage tissue were significantly decreased (P<0.01), while the percentage of chondrocytes with low Δψm, the average fluorescence intensity of ROS, and the expression of p62 protein in cartilage tissue were significantly increased (P<0.01). Compared to the model group, the acupotomy group showed no obvious defects on the surface of rabbit knee joint cartilage, relatively dense distribution of chondrocytes, increased autophagosomes, and relatively normal mitochondrial morphology. The fluorescence intensity of LC3B, PINK1 and Parkin, the expression of LC3Ⅱ/Ⅰ, PINK1 and Parkin proteins in cartilage tissue were significantly increased (P<0.01, P<0.05), while the percentage of chondrocytes with low Δψm, the average fluorescence intensity of ROS, and the expression of p62 protein in cartilage tissue were significantly decreased (P<0.01). CONCLUSION: Acupotomy may promote mitophagy by regulating the PINK1/Parkin pathway, thereby improving cartilage damage in rabbits with KOA.

[Effect of needle-knife on chondrocyte apoptosis of knee joint in rabbits with knee osteoarthritis based on CircSERPINE2-miR-1271-5P-ERG axis].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Effects on Autophagy of Moxibustion at Governor Vessel Acupoints in APP/PS1double-Transgenic Alzheimer's Disease Mice through the lncRNA Six3os1/miR-511-3p/AKT3 Molecular Axis.

Objective: To explore the effect and mechanism of moxibustion at acupoints of the governor vessel on lncRNA Six3os1 in amyloid precursor protein/presenilin1 (APP/PS1) double-transgenic Alzheimer's disease (AD) mice. Methods: Twenty-four specific pathogen-free and APP/PS1 double-transgenic male mice were randomly allocated into the AD model and moxibustion groups, with 12 cases in each group. Twelve syngeneic C57BL/6J mice were selected as the control group. Mice in the moxibustion group received aconite cake-separated moxibustion at the Baihui acupoint. Suspension moxibustion was applied at Fengfu and Dazhui for 15 minutes each day. All treatments were conducted over two weeks. Control and AD model mice were routinely fed without any intervention. Behavioral observation tests were conducted before and after the intervention. The autophagosome in the hippocampus was observed using transmission electron microscopy. Immunohistochemistry was performed to detect Aß1-42 expression. LC3B and P62 expressions were evaluated by immunofluorescence. The expression levels of the lncRNAs Six3os1, miR-511-3p, and AKT3 were detected by qRT-PCR. The differential expression of PI-3K, AKT3, mTOR, LC3B-II/I, and P62 proteins in the hippocampus was detected by western blot. The dual-luciferase assay was undertaken to examine the targeting relationships of the lncRNAs Six3os1, miR-511-3p, and AKT3. Results: Compared with the control group, the AD model showed higher escape latency in the Morris Water Maze and reduced autophagic vacuoles in the cytoplasm of hippocampal neurons (both p < 0.01). Compared with the control group, the AD model showed higher expression of Aß1-42, the lncRNAs Six3os1, PI-3K, mTOR, P62, and AKT3 protein (all p < 0.01); but lower mir-511-3p and LC3B (both p < 0.01). Compared with the AD model group, the moxibustion group had a shorter escape latency, more autophagic bubbles in the hippocampus, and lower expression of positive Aß1-42, the lncRNAs Six3os1, PI-3K, mTOR, P62, and AKT3 protein (all p < 0.01). In contrast, the levels of miR-511-3p and LC3B proteins were considerably increased in the moxibustion group compared to the AD model group (both p < 0.01). Based on the dual-luciferase assay, there was a targeting link among the lncRNAs Six3os1, miR-511-3p, and AKT3. Conclusion: Moxibustion at acupoints of the governor vessel can suppress the lncRNA Six3os1 expression, promote cell autophagy, accelerate Aß1-42 clearance and alleviate cognitive dysfunction of AD mediated by the PI3K/AKT/mTOR signaling pathway through the lncRNA Six3os1/miR-511-3p/AKT3 axis.