[Warm acupuncture stimulation improves cartilage damage and motor function by regulating JAK2/STAT3 signaling pathway in rabbits with knee osteoarthritis].

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

Hui Medicine Moxibustion Promotes the Absorption of Lumbar Disc Herniation and the Recovery of Motor Function in Rats through Fas/FasL Signaling Pathway.

Objectives: To study the resorption of the herniated lumbar disc (RHLD) and its mechanism in the SD rats of lumbar intervertebral disc herniation treated with Hui medicine moxibustion (HMM). Methods: Forty SD rats were randomly divided into four groups, normal group, lumbar disc herniation (LDH) group, HMM group, and antagonist (HMM+Met12) group, with 10 rats in each group. The rat model of LDH was prepared with the method of lumbar epidural emplacement of the caudal intervertebral disc. In the HMM group and HMM+Met12 groups, 4 weeks after modeling, HMM therapy was performed in the lumbar spine for 3 months with 1 time per day and 20 min each time, the samples were collected 8 weeks after the treatment. The histological degeneration was observed through HE staining, and the neovascularization of intervertebral disc tissues was detected by the expression of CD34 and vascular endothelial growth factor (VEGF). The apoptosis of nucleus pulpous cells was detected by TUNEL assay, and the activity of caspase-3, -8, and -9 and extracellular matrix enzymes was detected by western blotting. Results: HMM treatment significantly improved the behavioral ability of rats with LDH surgery. The morphological structure was obviously destroyed in the LDH group, but disc structure was significantly repaired in the HMM group, and mild structure alterations were observed in the HMM+Met12 group. Higher levels of CD34 and VEGF were detected in the HMM group indicating that neovascularization is formed. The expression level of FasL was significantly increased in the HMM group. The protein expression levels of cleaved-caspase-3, cleaved-caspase-8, and cleaved-caspase-9 in nucleus pulposus (NP) tissues were also elevated when treated with HMM, and the TUNEL staining showed the same results. The protein expression levels of matrix metalloproteinases- (MMP-) 1, MMP-2, MMP-3, MMP-13, and ADAMTS-4 were markedly promoted in the HMM group. Met12, a small peptide antagonist of FasL, significantly reduced the effects of HMM. Conclusion: HMM can promote the formation of neovascularization of lumbar intervertebral disc, support the apoptosis of NP cells through Fas/FasL signaling, and regulate the degradation of extracellular matrix enzyme, which then accelerates the absorption of lumbar intervertebral disc herniation and the recovery of motor function in rats.

Warming moxibustion attenuates inflammation and cartilage degradation in experimental rabbit knee osteoarthriti.

OBJECTIVE: To explore the molecular mechanism of warming moxibustion (WM) in knee osteoarthritis (KOA). METHODS: The knee joints of 40 New Zealand rabbits were placed in a plaster cast in an extended position to establish a KOA model. The animals were randomly divided into four groups: the control group, model group, WM group, and diclofenac (DF) group. Hematoxylin-eosin staining and the modified Mankin score were applied to evaluate the histopathological changes. Chondrocyte apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Western blotting and real-time quantitative polymerase chain reaction were performed to measure the expression of interleukin-1α (IL-1ß), prostaglandin E receptor 3 (PTGER3), a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-5 (ADAMTS-5), matrix metalloproteinase-13 (MMP-13), and C-terminal telopeptides of collagen type II (CTX-II) in cartilage tissues of the different groups. The concentrations of IL-1ß, PTGER3, and CTX-II in serum were detected by the enzyme-linked immunosorbent assay. RESULTS: Rabbits with KOA in the WM and DF groups showed significantly reduced cartilage erosion and Mankin scores, compared with the untreated rabbits. The number of TUNEL-positive cells observed in the WM group was much fewer than that in the model group. The expression of PTGER3, MMP-13, CTX-II IL-1ß, and ADAMTS-5 in cartilage tissues was remarkably downregulated following therapy with WM and DF. Moreover, a marked reduction was observed in the serum levels of IL-1ß, PTGER3, and CTX-II in the WM and DF groups. CONCLUSION: WM exerts favorable therapeutic effects on articular injuries of KOA by regulating the expression of inflammatory and cartilage degradation-related cytokines.

Effect of Warm Acupuncture Combined with Bone Marrow Mesenchymal Stem Cells Transplantation on Cartilage Tissue in Rabbit Knee Osteoarthritis.

The current study was designed to investigate the effect and underlying mechanism of warm acupuncture combined with bone marrow mesenchymal stem cells (BMSC) transplantation on cartilage tissue injury in rabbit knee osteoarthritis (KOA). In the study, 50 rabbits were randomly divided into 5 groups: blank group, KOA group, warm acupuncture group, BMSCs group, and warm acupuncture combined with BMSCs group. After warm acupuncture combined with BMSCs, the Modified Lequesne MG knee joint assessment scale was used to evaluate the degree of knee joint behavior, the Taiping Peng method generally observed the histomorphology changes of KOA rabbit cartilage, and hematoxylin-eosin staining, safranin O green staining, and toluidine blue staining were conducted to evaluate the extent of cartilage tissue pathology. Furthermore, transmission electron microscopy and TUNEL staining were used to observe cell apoptosis, and immunohistochemistry and qPCR analysis were used to detect the expression of apoptosis-related proteins and mRNA. Results showed that administration of warm acupuncture combined with BMSCs recovered the joint function and significantly decreased Lequesne MG score. The degree of cartilage tissue pathological damage has been improved, cartilage ultrastructure degeneration has recovered, peripheral blood vessels have mild edema, blood supply has gradually recovered, and even small amounts of red blood cells have appeared. In addition, warm acupuncture combined with BMSCs treatment suppressed chondrocyte apoptosis in rabbits with knee osteoarthritis by reduced TUNEL-positive chondrocytes and simultaneously reversed the mRNA expression of Bax, Bcl-2, and Caspase-3. These results indicate that warm acupuncture combined with BMSCs transplantation has a potential protective effect on rabbit KOA, which may be mediated by inhibiting chondrocyte apoptosis.

[Effect of warm needle moxibustion on morphological changes of cartilage and subchondral bone in knee osteoarthritis rabbits].

OBJECTIVE: To observe the effect of warm needle with moxibustion (WNM) on morphological changes of articular cartilage and subchondral bone tissues in knee osteoarthritis (KOA) rabbits, so as to explore whether WNM intervention can delay the development of KOA. METHODS: Forty male New Zealand rabbits were randomly divided into blank control, model, WNM and medication (Alendronate sodium) groups, with 10 rabbits in each group. A KOA model was established by immobilizing the right hind limb of rabbits with orthopedic casting tape for 6 weeks. The rabbits of the WNM group received WNM stimulation at "Neixiyan"(EX-LE4) "Waixiyan"(ST35) and "Heding"(EX-LE2) for 15 min, once a day for 4 weeks and those of the medication group received gavage of Alendronate sodium (150 µg·kg-1·d-1) once a day for 4 weeks. X-ray examination and magnetic resonance imaging (MRI) scanning were performed to observe the structure of the knee joints. Outcomes of X-ray examination were used to assess the degree of bone hyperplasia and joint space stenosis which were scaled according to Kellgren and Lawrence (K-L) standards, and those of MRI used to evaluate the degree of cartilage damage and bone marrow edema degrees which were respectively scored according to Recht scaling standards and semi-quantitative whole-organ MRI score (WORMS). Histopathological changes (degeneration degrees) of the articular cartilage were observed after H.E. staining, and given Mankin score. The ultrastructure of the cartilage surface and chondrocytes was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), respectively. RESULTS: Compared with the blank control group, the K-L grade, Recht grade, WORMS and Mankin score of the model group were significantly increased (P<0.05,P<0.01). After the intervention and in comparison with the model group, the K-L grade, Recht grade, WORMS and Mankin score of both WNM and medication groups were significantly decreased (P<0.05,P<0.01). Results of SEM showed severe defect, bulge and uneven surface of the cartilage with irregular growth and regeneration, and those of TEM showed degeneration and swelling of chondrocytes, margination of endonuclear chromatin, reduction in the number of organelles with disordered arrangement, extreme expansion of endoplasmic reticulum with detachment of the ribosome, unclear mitochondria, and disordered distribution of collagen fibers in the extracellular matrix in the model group, which was relatively milder in both the WNM and medication groups. Compared with the medication group, the Mankin score in the WNM group was evidently lower (P<0.05). CONCLUSION: WNM can effectively slow down the degeneration of cartilage and subchondral bone of KOA rabbits, delay the development of KOA.

Acidic leucine-rich nuclear phosphoprotein-32A expression contributes to adverse outcome in acute myeloid leukemia.

BACKGROUND: Acidic leucine-rich nuclear phosphoprotein-32A (ANP32A) is a novel regulator of histone H3 acetylation and promotes leukemogenesis in acute myeloid leukemia (AML). However, its prognostic value in AML remains unclear. METHODS: In this study, we evaluated the prognostic significance of ANP32A expression using two independent large cohorts of cytogenetically normal AML (CN-AML) patients. Multivariable analysis in CN-AML group was also presented. Based on the ANP32A expression, its related genes, dysregulation of pathways, interaction network analysis between microRNAs and target genes, as well as methylation analysis were performed to unveil the complex functions behind ANP32A. RESULTS: Here we demonstrated overexpression of ANP32A was notably associated with unfavorable outcome in two independent cohorts of CN-AML patients (OS: P=0.012, EFS: P=0.005, n=185; OS: P=0.041, n=232), as well as in European Leukemia Net (ELN) Intermediate-I group (OS: P=0.018, EFS: P=0.045, n=115), National Comprehensive Cancer Network (NCCN) Intermediate Risk AML group (OS: P=0.048, EFS: P=0.039, n=225), and non-M3 AML group (OS: P=0.034, EFS: P=0.011, n=435). Multivariable analysis further validated ANP32A as a high-risk factor in CN-AML group. Multi-omics analysis presented overexpression of ANP32A was associated with aberrant expression of oncogenes and tumor suppressor, up/down-regulation of metabolic and immune-related pathways, dysregulation of microRNAs, and hypomethylation on CpG island and 1st Exon regions. CONCLUSIONS: We proved ANP32A as a novel, potential unfavorable prognosticator and therapeutic target for AML.

Effect of stimulating acupoint Guanyuan (CV 4) on lower back pain by burning moxa heat for different time lengths: a randomized controlled clinical trial.

OBJECTIVE: To investigate the effect of different heat-stimulating time lengths on lower back pain. METHODS: Forty participants were randomly assigned to four groups of various heating time lengths. The short heating time length group (SL), moderate heating time length group (ML), and long heating time length group (LL) respectively received 15, 30, and 60 min of moxibustion therapy stimulating the acupoint Guanyuan (CV 4). The conventional acupuncture group (CA) received needle acupuncture treatment as a control group. The participants were treated continuously over a 2-week treatment period for a total of 10 sessions, with five sessions given per week. Participants were assessed weekly by blinded assessors,using the visual analogue scale (VAS) and Roland Morris Questionnaire (RMQ). RESULTS: The VAS and RMQ scores reduced in all four groups during treatment. There were significant differences in VAS scores (P < 0.01) and RMQ scores (P < 0.01) between before treatment and after 2 weeks of treatment in the LL group. After treatment, the LL group reported significantly lower VAS scores compared with the CA group, ML group, and SL group (P < 0.05). CONCLUSION: The long and moderate lengths of heat-stimulating time of 30 and 60 min may be more effective for relieving lower back pain than that of short stimulating time lengths.

Effect of acupuncture anesthesia on acne vulgaris of pricking-bloodletting cupping: a single-blind randomized clinical trail.

OBJECTIVE: To evaluate the effect on acne vulgaris of pricking-bloodletting cupping at Dazhui (GV 14) under acupuncture anesthesia, and establish whether providing anesthesia to the treatment area by manipulating Hegu (LI 4) and Quchi (LI 11) might have an additional therapeutic benefit. METHODS: Thirty-eight patients were recruited and randomized into a control group and an intervention group with a single-blind (observer-blind) method. The control group was treated by pricking-bloodletting cupping at Dazhui (GV 14)-and the studied group by pricking-bloodletting cupping at Dazhui (GV 14) under acupuncture anesthesia at Hegu (LI 4) and Quchi (LI 11). Both groups were treated twice weekly for 6 weeks. The analgesic and therapeutic effects of acupuncture were evaluated on a visual analog scale (VAS) and global acne grading system (GAGS), respectively. RESULTS: There were differences in the VAS scores of pain on pricking and in the pricked area, and the duration of pain between the groups. After 12 treatments, there was a significant reduction in GAGS scores from baseline in both groups, but there was no significant difference between the groups. CONCLUSION: Acupuncture anesthesia at Hegu (LI 4) and Quchi (LI 11) is an effective means of alleviating the pain of pricking-bloodletting cupping and reducing the duration of pain in the treatment area. Pricking-bloodletting cupping at Dazhui (GV 14) improves the skin lesions of patients with moderate acne vulgaris, but acupuncture anesthesia does not appear to have an additional therapeutic effect.