| Objective: To explore the effect of cast external fixation on the formation of knee extension contracture in rabbits and the role of skeletal muscle autophagy in the formation of knee joint contracture in the first part.To investigate the therapeutic effect of low-frequency electrical stimulation on rabbit knee extension contracture and its relationship with skeletal muscle autophagy in the second part.Methods: This experiment consists of two parts.In the first part of the study,24 rabbits were randomly divided into four groups,each with 6 rabbits.Control 1 group(Ctrl1group,no fixation)was given free activities for 6 weeks.2 weeks of immobilization group(Ⅰ-2 group),4 weeks of immobilization group(Ⅰ-4 group)and 6 weeks of immobilization group(Ⅰ-6 group)were fixed the left hind knee joint of rabbits in extension position for 2 weeks,4 weeks and 6 weeks respectively.The rabbits were euthanized at the end of immobilization,and the left hip joint was dislocated so that the left hind limb was completely separated from the trunk.The range of motion(ROM)of the knee joint of the left hind limb was measured by a joint motion meter,and the cross-sectional area of skeletal muscle(CSA)was measured via HE staining.The expression of autophagy-related proteins p-m TOR,Atg7,p62 and LC3B-Ⅱ in skeletal muscle was detected by Western Blot,looking for the time point at which skeletal muscle autophagy is highest.And the expression of LC3 B in skeletal muscle was detected by immunofluorescence.Mediation analysis explored the role of skeletal muscle autophagy in the formation of knee contracture.In the second part of the experiment,24 rabbits were also randomly divided into four groups,each group with 6rabbits.Control 2 group(Ctrl2 group,no fixation)was given free activities for 7 weeks.In the simple electrical stimulation group(ESG group),3 weeks of low-frequency electrical stimulation was given after 4 weeks of free activity.The left knee joint of natural recovery group(NRG group)and electrical stimulation treatment group(ESTG group)were fixed in the extension position for 4 weeks.The NRG group recovered naturally for 3 weeks after 4 weeks of fixation;the ESTG group was given 3 weeks of low-frequency electrical stimulation after 4 weeks of fixation.The rabbits were euthanized at the end of the intervention.The left hip joint was dislocated so that the left hind limb was completely separated from the trunk.The range of motion(ROM)of the knee joint of the left hind limb was measured by a joint motion meter.HE staining was used to measure skeletal muscle cross-sectional area(CSA).The expression of autophagy-related proteins p-m TOR,Atg7,p62 and LC3B-Ⅱ in skeletal muscle was detected by Western Blot.Mediation analysis was conducted to explore the therapeutic effect of low frequency electrical stimulation on extension knee contracture and its mechanism in rabbits.Results: Part 1 found that knee joint ROM and rectus femoris CSA gradually decreased with the extension of fixation time.Compared with the Ctrl1 group,the differences were statistically significant(P<0.05).Compared with the Ctrl1 group,the expression of autophagy-related proteins p-m TOR,p62 and LC3B-Ⅱ in the Ⅰ-4 group increased most significantly(P<0.05).Compared with the Ctrl1 group,the expression level of Atg7 protein in the Ⅰ-4 group was also increased significantly(P<0.01).The result of mediation analysis showed that the increased expression of skeletal muscle autophagy-related protein played a partial mediation role in the formation of joint contractures caused by fixation.The second part of this experiment found that the ROM of electrical stimulation treatment group(ESTG group)was improved more significantly than natural recovery group(NRG group)(P<0.01);the CSA of ESTG group was also increased significantly compared with the NRG group(P<0.05).In addition,the expression levels of p-m TOR,Atg7,p62 and LC3B-Ⅱ in NRG group were higher than those of the control 2 group(Ctrl2 group)and the electrical stimulation group(ESG group)(P<0.01).The expression levels of autophagy-related proteins such as p-m TOR,p62 and LC3B-Ⅱ in ESTG group were lower than those of the NRG group(P<0.01).The expression of Atg7 protein in the ESTG group was also lower than that in the NRG group(P<0.05).The result of mediation analysis showed that the decreased expression of skeletal muscle autophagy-related protein played a partial mediation role in the process of low-frequency electrical stimulation improving joint contracture.Conclusions: Knee extension fixation could induce rabbit skeletal muscle atrophy,knee joint contracture and increased expression of skeletal muscle autophagy-related protein.The increase of skeletal muscle autophagy-related protein expression is related to the formation of rabbit knee joint contracture.Inhibiting the excessive activation of skeletal muscle autophagy may be one of the mechanisms by which low-frequency electrical stimulation quadriceps femoris exerts a therapeutic effect. |
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