Effects of hepatocyte-derived exosomes from mice on knee cartilage of model mice with knee osteoarthritis:a softening liver and nourishing tendons theory-based experimental study
Objective:To explore the effects of hepatocyte-derived exosomes(HDEs)from mice on knee cartilage of model mice with knee osteoarthritis(KOA)based on softening liver and nourishing tendons theory.Methods:The exosomes were isolated and extracted from the livers of young(3-month-old)and aged(22-month-old)male C57BL/6 mice by using ultracentrifugation.Forty-eight 7-week-old specific pathogen free(SPF)-grade male C57BL/6J mice were randomized into KOA group,Young-EVs group,Aged-EVs group and Sham group,12 ones in each group.The mice in KOA group,Young-EVs group and Aged-EVs group were subjected to anterior cruciate ligament(ACL)transection for inducing KOA;while the ones in Sham group only underwent surgery,but without transecting ACL.At postoperative day 14 after the modeling,the mice in Young-EVs group and Aged-EVs group were intervened by knee intra-articular injection of 10 μL HDEs ex-tracted from young and aged mice,respectively,two times a week for consecutive 4 weeks;while the ones in Sham group and KOA group with 10 μL phosphate buffered saline(PBS)solution.After the end of intervention,the knee joints of the modeling side in mice of each group were checked by X-ray scanning,and were evaluated by Kellgren-Lawrence(K-L)grading system.After the end of X-ray examination,the knees were harvested from mice of each group,and the knee tissue sections were stained with hematoxylin-eosin(HE)and safranin O-fast green(SO-FG)for observing the histopathological changes;meanwhile,the degree of knee articular cartilage degeneration was evalua-ted by using Osteoarthritis Research Society International(OARSI)osteoarthritis cartilage histopathology assessment system and Mankin sco-ring system.Furthermore,the expression levels of collagen Ⅱ(COL Ⅱ),aggrecan(ACAN),matrix metalloproteinase(MMP)13 and a disin-tegrin and metalloproteinase with thrombospondin motifs(ADAMTS)5 in knee cartilage tissues were detected by using immunohistochemis-try(IHC).Results:①The results of X-ray examination showed that the mice in Sham group presented with normal knee joint space,smooth articular surface,and intact cartilage,and no osteophyte was observed at the margin of knee joint.The K-L classification belonged to grade 0.The changes,manifesting as rough articular surface,partially defective cartilage and definite osteophytes at the margin of knee joint,were observed,while,the marked knee joint space narrowing(JSN)were unobserved in mice of KOA group.The K-L classification belonged to grade Ⅱ.The slight JSN,slightly rough articular surface,defective cartilage(decreased compared to KOA group),and small osteophytes at the margin of knee joint were observed in mice of Young-EVs group.The K-L classification belonged to grade Ⅰ.Compared to KOA group,the changes,including knee joint space,articular surface roughness,cartilage defects,and the number of free bodies within the joint,were more severe in mice of Aged-EVs group.The K-L classification belonged to grade Ⅲ.②There was statistical difference in OARSI score and Mankin score between the 4 groups(OARSI score:0.417±0.376,4.500±0.632,2.417±0.492,5.417±0.492 points,F=116.800,P=0.000;Mankin score:1.083±0.665,7.250±0.880,3.583±0.917,8.833±0.683 points,F=117.100,P=0.000).The OARSI score and Mankin score were higher in KOA group,Young-EVs group and Aged-EVs group compared to Sham group(P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000),and were higher in KOA group compared to Young-EVs group(P=0.000,P=0.000),and were highest in Aged-EVs group(P=0.025,P=0.000;P=0.015,P=0.000).③There was statistical difference in the posi-tive expression area ratios of COL Ⅱ and ACAN in knee cartilage between the 4 groups(COL Ⅱ:40.050±2.324,26.083±1.119,23.317±2.599,16.717±2.479％,F=118.500,P=0.000;ACAN:51.500±3.191,17.700±3.415,19.383±3.924,12.300± 1.908％,F=185.500,P=0.000).The positive expression area ratios of COL Ⅱ and ACAN were lower in KOA group,Young-EVs group and Aged-EVs group compared to Sham group(P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000),and were lower in Aged-EVs group compared to KOA group and Young-EVs group(P=0.000,P=0.000;P=0.039,P=0.005),while,the comparison be-tween Young-EVs group and KOA group revealed no significant differences(P=0.167,P=0.799).There was statistical difference in the positive expression area ratios of MMP13 and ADAMTS5 in knee cartilage between the 4 groups(MMP13:11.733±2.191,46.150± 6.237,34.417±5.027,57.233±4.049％,F=106.600,P=0.000;ADAMTS5:7.950±1.344,30.533±6.184,22.083±3.418,39.317±5.606％,F=51.450,P=0.000).The positive expression area ratios of MMP13 and ADAMTS5 were higher in KOA group,Young-EVs group and Aged-EVs group compared to Sham group(P=0.000,P=0.000,P=0.000;P=0.000,P=0.000,P=0.000),and were higher in KOA group compared to Young-EVs group(P=0.002,P=0.021),and were highest in Aged-EVs group(P=0.003,P=0.000;P=0.016,P=0.000).Conclusion:The mice HDEs may be involved in the synthesis and catabolic reactions of chondrocytes,among which HDEs from the aged mice can accelerate the degradation of extracellular matrix and reduce the synthesis of AC AN,thereby ex-acerbating the cartilage degeneration in KOA mice;whereas the ones from the young mice can inhibit the degradation of cartilage extracellu-lar matrix,thus exerting the effect of protecting cartilage in KOA mice.
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