OBJECTIVE To investigate the mechanism by which the traditional Chinese medicine formula Xibining Ⅱ modulates cold-stimulus pain sensitivity in rats with cold-damp obstruction-type knee osteoarthritis （KOA） based on the SET domain bifurcated histone lysine methyltransferase 2 （SETDB2）/histone H3 lysine 9 trimethylation （H3K9me3） signaling axis. METHODS Fifty SD rats were randomly divided into control group （intragastric administration and intrathecal injection of equal volumes of normal saline）， model group （intragastric administration and intrathecal injection of equal volumes of normal saline）， Xibining Ⅱ low- and high-dose groups （4， 8 g/kg Xibining Ⅱ， intragastric administration）， and high-dose of Xibining Ⅱ+small interfering RNA （siRNA） group （8 g/kg of Xibining Ⅱ via intragastric administration and intrathecal injection of SETDB2 siRNA at 0.2 mmol/L， 20 μL per rat）， with 10 rats in each group. Except for the control group， cold-damp obstruction-type KOA model was induced in other groups. Drug administration commenced 14 days post-modeling and continued for 28 days. Following the final administration， the following were assessed： behavioral changes in cold-stimulation pain sensitivity， histopathological changes in the articular cartilage of the knee joint， the contents of inflammatory factors [tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）] and pain mediators [calcitonin gene-related peptide （CGRP）， nerve growth factor （NGF）]， as well as the expressions of SETDB2/H3K9me3 signaling axis，inflammatory factors and pain mediators related proteins and mRNAs in dorsal root ganglion （DRG） tissue. RESULTS After 28 days of drug administration， compared with the model group， Xibining Ⅱ low- and high-dose groups exhibited significantly prolonged cold-stimulus paw withdrawal latency （P＜0.05）； the number of positive responses in the acetone low-temperature test was significantly reduced （P＜0.05）； Mankin score and the Osteoarthritis Research Society International score for knee joint tissue， as well as the levels of inflammatory factors and pain mediators in the serum and their expression in DRG tissue were all significantly decreased （P＜0.05）； the protein expressions of SETDB2 and H3K9me3 in DRG tissue were significantly increased （P＜0.05）. Intrathecal injection of SETDB2 siRNA reversed the above effects of high-dose of Xibining Ⅱ （P＜0.05）. CONCLUSIONS Xibining Ⅱ may alleviate inflammatory and pain responses by activating the SETDB2/H3K9me3 signaling axis， ultimately improving cold-stimulus pain sensitivity in rats with cold-damp obstruction-type KOA.

ObjectiveTo establish and evaluate different disease-syndrome combination mice models of knee osteoarthritis （KOA） with cold-dampness obstruction syndrome， and to identify the optimal modeling method. MethodsEighty mice were randomly divided into a control group， a treadmill plus climate chamber group， an anterior cruciate ligament transection （ACLT） plus climate chamber group， an ACLT group， a destabilization of the medial meniscus （DMM） plus climate chamber group， a DMM group， an intra-articular injection plus climate chamber group， and an intra-articular injection group， with 10 mice in each group. Mice in the ACLT group underwent surgical transection of the knee anterior cruciate ligament to establish the model， while those in the DMM underwent surgical transection of the medial meniscal tibial ligament of the knee， and those in the intra-articular injection group were modeled by an intra-articular injection of 50 μl of normal saline containing 1mg of monosodium iodoacetate （MIA） into the medial side of the patellar ligament. Mice in the ACLT plus climate chamber， DMM plus climate chamber， and intra-articular injection plus climate chamber groups were placed in an artificial climate chamber （relative humidity 60%， temperature 4 ℃， wind speed 6 m/s） simultaneously with aforementioned interventions， once daily for 2 h each time， for 14 consecutive days. Mice in the control group underwent sham surgery， involving only the incision of the skin and subcutaneous tissue at the knee joint to expose the articular cavity， followed by layer-by-layer suturing. Mice in the treadmill plus climate chamber group were placed on an animal treadmill starting on the 4th day after the sham operation， undergoing high-intensity treadmill exercise at a speed of 20 m/min， an inclination of 5°， for 30min per session， 5 times per week for 28 consecutive days， while being housed in the artificial climate chamber. Cold pain thresholds of the mice in each group were compared using the cold plate test and the acetone low-temperature test before and after modeling. Following modeling， knee joint pathological changes were observed using HE and Safranin O-Fast Green staining， and quantified using Mankin and Osteoarthritis Research Society International （OARSI） scores. Serum levels of tumor necrosis factor-α （TNF-α） and interleukin-1β （IL-1β） were measured by ELISA. Protein and mRNA expression levels of pain-related markers， including calcitonin gene-related peptide （CGRP）， protein gene product 9.5 （PGP9.5）， and cellular proto-oncogene Fos （c-Fos）， and the cold hyperalgesia-related markers including transient receptor potential ankyrin 1 （TRPA1） and transient receptor potential melastatin 8 （TRPM8） in the dorsal root ganglion （DRG） tissues were detected by Western Blot and qPCR. TRPA1 and TRPM8 fluorescent intensity in the DRG was detected by immunofluorescence assay. ResultsCompared to the control group， the remaining groups showed decreased cold pain thresholds in the cold plate tese， increased cold pain thresholds in the acetone low-temperature test， elevated Mankin and OARSI scores， and increased serum TNF-α and IL-1β levels， upregulated protein and mRNA expression levels of CGRP， PGP9.5， c-Fos， TRPA1， and TRPM8 in the DRG， and increased immunofluorescence intensities of TRPA1 and TRPM8 in the DRG tissue （P＜0.05 or P＜0.01）， along with severe pathological damage and cartilage degeneration in the knee joints. Compared to the ACLT， DMM， and intra-articular injection groups， the ACLT plus climate chamber， DMM plus climate chamber， and intra-articular injection plus climate chamber groups exhibited more significant changes in all the aforementioned indicators （P＜0.01）. Furthermore， changes in the aforementioned indicators were more pronounced in the ACLT plus climate chamber group compared to the Treadmill plus climate chamber group， DMM plus climate chamber and intra-articular injection plus climate chamber group （P＜0.01）， exhibiting the most severe degree of pathological damage and cartilage degeneration. ConclusionThe combination of an artificial climate chamber can successfully establish a disease-syndrome combined animal model of KOA with cold-damp obstruction syndrome， with the ACLT combined with the artificial climate chamber being the optimal modeling approach.

ObjectiveTo establish and evaluate different disease-syndrome combination mice models of knee osteoarthritis （KOA） with cold-dampness obstruction syndrome， and to identify the optimal modeling method. MethodsEighty mice were randomly divided into a control group， a treadmill plus climate chamber group， an anterior cruciate ligament transection （ACLT） plus climate chamber group， an ACLT group， a destabilization of the medial meniscus （DMM） plus climate chamber group， a DMM group， an intra-articular injection plus climate chamber group， and an intra-articular injection group， with 10 mice in each group. Mice in the ACLT group underwent surgical transection of the knee anterior cruciate ligament to establish the model， while those in the DMM underwent surgical transection of the medial meniscal tibial ligament of the knee， and those in the intra-articular injection group were modeled by an intra-articular injection of 50 μl of normal saline containing 1mg of monosodium iodoacetate （MIA） into the medial side of the patellar ligament. Mice in the ACLT plus climate chamber， DMM plus climate chamber， and intra-articular injection plus climate chamber groups were placed in an artificial climate chamber （relative humidity 60%， temperature 4 ℃， wind speed 6 m/s） simultaneously with aforementioned interventions， once daily for 2 h each time， for 14 consecutive days. Mice in the control group underwent sham surgery， involving only the incision of the skin and subcutaneous tissue at the knee joint to expose the articular cavity， followed by layer-by-layer suturing. Mice in the treadmill plus climate chamber group were placed on an animal treadmill starting on the 4th day after the sham operation， undergoing high-intensity treadmill exercise at a speed of 20 m/min， an inclination of 5°， for 30min per session， 5 times per week for 28 consecutive days， while being housed in the artificial climate chamber. Cold pain thresholds of the mice in each group were compared using the cold plate test and the acetone low-temperature test before and after modeling. Following modeling， knee joint pathological changes were observed using HE and Safranin O-Fast Green staining， and quantified using Mankin and Osteoarthritis Research Society International （OARSI） scores. Serum levels of tumor necrosis factor-α （TNF-α） and interleukin-1β （IL-1β） were measured by ELISA. Protein and mRNA expression levels of pain-related markers， including calcitonin gene-related peptide （CGRP）， protein gene product 9.5 （PGP9.5）， and cellular proto-oncogene Fos （c-Fos）， and the cold hyperalgesia-related markers including transient receptor potential ankyrin 1 （TRPA1） and transient receptor potential melastatin 8 （TRPM8） in the dorsal root ganglion （DRG） tissues were detected by Western Blot and qPCR. TRPA1 and TRPM8 fluorescent intensity in the DRG was detected by immunofluorescence assay. ResultsCompared to the control group， the remaining groups showed decreased cold pain thresholds in the cold plate tese， increased cold pain thresholds in the acetone low-temperature test， elevated Mankin and OARSI scores， and increased serum TNF-α and IL-1β levels， upregulated protein and mRNA expression levels of CGRP， PGP9.5， c-Fos， TRPA1， and TRPM8 in the DRG， and increased immunofluorescence intensities of TRPA1 and TRPM8 in the DRG tissue （P＜0.05 or P＜0.01）， along with severe pathological damage and cartilage degeneration in the knee joints. Compared to the ACLT， DMM， and intra-articular injection groups， the ACLT plus climate chamber， DMM plus climate chamber， and intra-articular injection plus climate chamber groups exhibited more significant changes in all the aforementioned indicators （P＜0.01）. Furthermore， changes in the aforementioned indicators were more pronounced in the ACLT plus climate chamber group compared to the Treadmill plus climate chamber group， DMM plus climate chamber and intra-articular injection plus climate chamber group （P＜0.01）， exhibiting the most severe degree of pathological damage and cartilage degeneration. ConclusionThe combination of an artificial climate chamber can successfully establish a disease-syndrome combined animal model of KOA with cold-damp obstruction syndrome， with the ACLT combined with the artificial climate chamber being the optimal modeling approach.

AIM:To investigate whether Xibining Ⅱ(XBN Ⅱ)attenuates cartilage damage in rats with knee osteoarthritis(KOA)by modulating glycolysis via the AMP-activated protein kinase(AMPK)/peroxisome proliferator-acti-vated receptor γ coactivator 1α(PGC1α)signaling pathway.METHODS:Thirty-two SD rats were randomly divided into sham group,KOA group,XBN Ⅱ group and metformin(AMPK activator)group,with 8 rats in each group.The rats in KOA group were subjected to the anterior cruciate ligament transection procedure to establish the KOA model.Starting from the 14th day after modeling,the rats in XBN Ⅱ group received a daily dose of XBN Ⅱ via gavage once a day,and those in metformin group were administered metformin via intraperitoneal injection once a day for 4 weeks.Subsequently,the histopathological changes of the cartilage were examined by HE and safranin O-fast green staining with matching Mankin and OARSI scores.The protein levels of phosphorylated AMPK(p-AMPK)and PGC1α in cartilage were quanti-fied through immunohistochemistry.In addition,RT-qPCR and Western blot were conducted to measure the mRNA and protein expression levels of glycolysis-related factors,including glucose transporter 1,hexokinase 2 and lactate dehydroge-nase A,biomarkers related to cartilage synthesis and catabolism,such as collagen type Ⅱ,aggrecan,matrix metallopro-teinase 13 and a disintegrin and metalloproteinase with thrombospondin motifs 5,and AMPK/PGC1α signaling pathway-re-lated indicators.RESULTS:Lactate levels in cartilage and serum were higher in KOA group compared with sham group(P<0.05).Similarly,the cartilage in KOA group exhibited significant surface abrasion and structural damage,with faint-stained matrix and significantly higher Mankin and OARSI scores compared with sham group(P<0.05).Further analysis revealed significant decreases in the mRNA and protein expression levels of factors related to cartilage anabolism and AMPK/PGC1α signaling pathway in KOA group compared with sham group(P<0.05).In contrast,there were marked in-creases in the mRNA and protein expression levels of factors related to cartilage catabolism and glycolysis(P<0.05).No-tably,XBN Ⅱ and metformin treatments significantly improved the cartilage morphology,reduced Mankin and OARSI scores,and reversed the changes in mRNA and protein levels of the aforementioned indexes(P<0.05).CONCLU-SION:Treatment with XBN Ⅱ can alleviate cartilage damage in KOA rats by inhibiting glycolysis,through a mechanism involving activation of the AMPK/PGC1α signaling pathway.

AIM:To investigate whether Xibining Ⅱ(XBN Ⅱ)attenuates cartilage damage in rats with knee osteoarthritis(KOA)by modulating glycolysis via the AMP-activated protein kinase(AMPK)/peroxisome proliferator-acti-vated receptor γ coactivator 1α(PGC1α)signaling pathway.METHODS:Thirty-two SD rats were randomly divided into sham group,KOA group,XBN Ⅱ group and metformin(AMPK activator)group,with 8 rats in each group.The rats in KOA group were subjected to the anterior cruciate ligament transection procedure to establish the KOA model.Starting from the 14th day after modeling,the rats in XBN Ⅱ group received a daily dose of XBN Ⅱ via gavage once a day,and those in metformin group were administered metformin via intraperitoneal injection once a day for 4 weeks.Subsequently,the histopathological changes of the cartilage were examined by HE and safranin O-fast green staining with matching Mankin and OARSI scores.The protein levels of phosphorylated AMPK(p-AMPK)and PGC1α in cartilage were quanti-fied through immunohistochemistry.In addition,RT-qPCR and Western blot were conducted to measure the mRNA and protein expression levels of glycolysis-related factors,including glucose transporter 1,hexokinase 2 and lactate dehydroge-nase A,biomarkers related to cartilage synthesis and catabolism,such as collagen type Ⅱ,aggrecan,matrix metallopro-teinase 13 and a disintegrin and metalloproteinase with thrombospondin motifs 5,and AMPK/PGC1α signaling pathway-re-lated indicators.RESULTS:Lactate levels in cartilage and serum were higher in KOA group compared with sham group(P<0.05).Similarly,the cartilage in KOA group exhibited significant surface abrasion and structural damage,with faint-stained matrix and significantly higher Mankin and OARSI scores compared with sham group(P<0.05).Further analysis revealed significant decreases in the mRNA and protein expression levels of factors related to cartilage anabolism and AMPK/PGC1α signaling pathway in KOA group compared with sham group(P<0.05).In contrast,there were marked in-creases in the mRNA and protein expression levels of factors related to cartilage catabolism and glycolysis(P<0.05).No-tably,XBN Ⅱ and metformin treatments significantly improved the cartilage morphology,reduced Mankin and OARSI scores,and reversed the changes in mRNA and protein levels of the aforementioned indexes(P<0.05).CONCLU-SION:Treatment with XBN Ⅱ can alleviate cartilage damage in KOA rats by inhibiting glycolysis,through a mechanism involving activation of the AMPK/PGC1α signaling pathway.