高寒环境通过诱导骨骼肌萎缩与纤维化协同加重骨关节炎病理进程OA

Objective To investigate the impact of a high-altitude and cold environment on skeletal muscle function and the pathological processes of osteoarthritis(OA),to elucidate the interaction mechanisms between skeletal muscle atrophy,fibrosis,and joint degeneration under cold condition,aiming to found a theoretical basis for the health protection of the musculoskeletal system in military personnel stationed in high-altitude cold regions.Methods Destabilization of the medial meniscus(DMM)was established on C57BL/6 mice(11 weeks old)by cutting the attaching point at the anterior intercondylar region where anterior cruciate ligament blends with the medial meniscus.Forty male C57BL/6 mice(11 weeks old)were stratified by 2 postoperative time points(8 and 12 weeks).At each time point,the mice were divided randomly divided into 4 groups(n=5):22℃sham operation(22℃Sham),4℃sham operation(4℃Sham),22℃DMM,and 4℃DMM.From 2 weeks after DMM modelling,the 4℃Sham and 4℃DMM groups were placed in a cold chamber[4℃,humidity(50±5)%]for 12 h per day until the respective endpoint,meanwhile,the 22℃Sham and 22℃DMM groups were kept at room temperature.At 8 and 12 weeks after intervention,the following assessments were carried out:① Behavioral tests:Gait parameters were quantified using the CatWalk system;Limb load-bearing capacity was evaluated with bipedal balance test;Spontaneous activity distance and time were recorded in the open field test;Twitch force and tetanic force in the quadriceps were measured,and the force-frequency curve was plotted.② Histopathological analysis:Quadriceps and knee joint tissues were harvested,embedded in paraffin,sectioned and stained with HE and safranin O-fast green to assess synovial inflammatory cell infiltration,cartilage surface integrity,and OARSI score from Osteoarthritis Research Society International System.Additionally,Masson's trichrome staining was applied to observe collagen deposition in muscle fibers.③ Molecular detection:Immunofluorescence assay for dystrophin antibody was used to label muscle fiber boundaries and calculate cross-sectional area;Immunohistochemistry assay was employed to determine the expression levels of IL-1β and TNF-α in synovial tissue.Results ① Progressive deterioration of behavioral function:Compared with the 22℃DMM group,the 4℃DMM group exhibited significantly exacerbated functional impairments at both 8 and 12 weeks after intervention,in a time-dependent worsening trend.a.Loss of gait coordination:At 8 weeks,the relative stance phase time of the right hindlimb was shorten by 0.113(P<0.001),print area was reduced by 0.102(P<0.05),swing phase time was extended by 0.115(P<0.05),and single-limb stand time was prolonged by 0.146(P<0.01).These abnormalities still existed at 12 weeks,with the difference of print area expanding to 0.154(P<0.05)and that of swing phase time maintaining an elevated magnitude of 0.113(P<0.05).b.Decline in weight-bearing capacity:Percentage of right paw weight-bearing was decreased by 2.777%at 8 weeks(P<0.001),with the deficit further expanding to 5.156%at 12 weeks(P<0.000 1).c.Suppression of spontaneous activity:Total locomotor distance was decreased by 1 069 cm at 8 weeks(P<0.000 1)and remained reduced by 429.3 cm at 12 weeks(P<0.05).② Exacerbation of joint histopathological damage:Cold exposure combined with DMM surgery significantly aggravated synovial inflammation and cartilage destruction.a.Synovial pathology:The 4℃DMM group displayed increased synovial pathological score of 2.133 at 8 weeks(P<0.01)and then 2.833 at 12 weeks(P<0.001)relative to the 22℃DMM group,indicating a sustained inflammatory response.b.Cartilage degeneration:The 4℃DMM group showed the maximum score of OARSI elevated by 1.000(P<0.05)and total score by 2.000(P<0.01)at 8 weeks,while,at 12 weeks,the maximum score remained elevated by 0.750,and the total score maintained a differential trend of 2.000.c.Upregulation of pro-inflammatory factors:The synovial expression area of IL-1β in the 4℃DMM group was increased by 1.047%(P<0.05)and 1.892%(P<0.05)at 8 and 12 weeks,respectively,and that of TNF-α expression was increased by 3.850%(P<0.000 1)and 3.806%(P<0.000 1),separately at the same time points.③ Composite impairment of skeletal muscle structure and contractile function:Cold intervention induced triple pathological changes in quadriceps encompassing atrophy,mechanical performance decline,and fibrotic remodeling.a.Depletion of muscular force reserve:Twitch force was decreased by 34.77 mN·m/kg(P<0.01)and 52.87 mN·m/kg(P<0.05)at 8 and 12 weeks,respectively;Tetanus force was reduced by 115.0 mN·m/kg(P<0.05)and 138.3 mN·m/kg(P<0.000 1)at the same time points,with a downward shift of the force-frequency curve.b.Muscle atrophy:The mass ratio of quadriceps was decreased by 0.076%(P<0.05)and 0.102%(P<0.000 1),and the muscle fiber cross-sectional area(CSA)was reduced by 261.3 μm²(P<0.05)and 233.5 μm²(P<0.01)at 8 and 12 weeks,respectively,accompanied with an increased proportion of small-diameter fibers.c.Fibrotic deposition:Relative collagen fiber area in muscle tissue was increased by 4.072%(P<0.01)and 2.238%(P<0.05)at 8 and 12 weeks,respectively,suggesting abnormal extracellular matrix remodeling.Conclusion High-altitude cold environment not only directly aggravates synovial inflammation and cartilage degeneration in OA but also further compromises joint structure and function by inducing quadriceps atrophy,reduced muscle strength,and fibrosis.