| Background:At the moment, the studies on the muscle atrophy indicated that the direct result of skeletal muscle atrophy is reducing of protein synthesis and increasing its degradation, and the most important change is the protein degradation. The preview found in our laboratory was demonstrated that the intracellular Ca2+ overloading in skeletal muscle under disuse, and then activated the pathway of protein degradation. Other studies showed that the apoptosis pathway was closely related to the intracellular Ca2+ overloading, which Bax and Bcl-2 were apoptosis-related factors to promote or restrain apoptotic genes respectively in animals’ tissues.Objective:1. To study the effect of Ca2+ in disuse status rats and ground squirrels and its relationship with protein degradation, and then analyse the results of experimental to found the mechanisms to anti-muscle atrophy in hibernation inactivity of ground squirrels;2. To analyse the changes of apoptosis-related factors Bax and Bcl-2 and its ratio in the expression of gene and protein level. Besides, we also discussed the relationship between apoptosis and muscle atrophy and discuss the possible mechanism of anti-muscle atrophy and apoptosis of ground squirrels in hibernating period.Methods:1. The hindlimb-unloading model was by referring to the tail-suspension rats;2. The Ca2+ concentration in living cells in EDL fiber was observed via Ca2+ fluorescence intensity, which was determined by confocal laser scanning method;3. To determine the interrelated apoptosis gene expression level-Bax and Bcl-2 mRNA in gene level of EDL of body by real-time quantitative PCR method;4. To determine its relative expression of Bax and Bcl-2 and calculate its ratio in protein level by Western Blot technique.Results:1. In rats, the concentration of Ca2+ in EDL fiber increased by 68.6%(P＜0.05) in HLU+W group; Compared with HLU+W group, the concentration of Ca2+ in EDL fiber decreased by 46.2%(P＜0.05) in HLU+T group;2. In ground squirrels, the concentration of EDL fiber Ca2+ in IB-A group increased by 60.0%(P<0.05) compared with Pre-H group; compared with H group, the concentration of EDL fiber Ca2+ in IB-A and Post-H increased by 68.9% (P<0.05) and 41.8% (P<0.05) respectively; the concentration of Ca2+ in H group is no significance difference compared with Pre-H group;3. In gene expression level, in EDL and SOL, the ratio of Bax/Bcl-2 in HLU+W group increased by 30.9% (P<0.05) and 35.3% (P<0.05); the ratio of Bax/Bcl-2 in Post-H ground squirrel reduced by 28.3% (P<0.01) and 17.7% (P< 0.05);4 In protein expression level, in ground squirrels skeletal muscle (EDL and SOL), the ratio of Bax/Bcl-2 in IB-A group increased by 82.5% (P＜0.05) and 26.9% (P＜0.05) compared with H group, in SOL, the ratio of Bax/Bcl-2 in H group reduced by 23.5% (P<0.05) compared with Pre-H group.Conclusions:1. After 14d hibernation-unloading, the Ca2+ concentration in rats EDL increased significantly, which would promote the calpains transfer to the cell membrane and degrade some proteins and lead to skeletal muscle atrophy; In HLU+T group, the significantly decreased Ca2+ concentration may be due to Ligustrazine that can restrain calcium channel on cell membrane, and impede the calcium ions into the cytoplasm, which showing that Ligustrazine can anti-muscle atrophy;2. There is not appearing calcium overload phenomenon in hibernating ground squirrel, which is accordance with no muscle atrophy in hibernation. The Ca2+ concentration significantly increased in IB-A group, which shows that cytosolic calcium overload is an orderly process, when the Ca2+ concentration over body regulation threshold, will cause the ground squirrel arousal to response to this undesirable effect, which may also relation to energetics related strategies of the squirrel during hibernation;3. In gene expression level, after 14d hibernation-unloading of rats, the Bax/Bcl-2 ratio significantly increased, which shows that apoptosis phenomenon during the disuse period was occurred and finally induced muscle atrophy. But it recovered to the original level in HLU+T group, which shows that the Ligustrazine can against the disuse muscle atrophy. In Post-H ground squirrel group, the BaxlBcl-2 ratio significantly decreased, which shows that the ability of Bcl-2 has relatively strengthened;4. In protein expression level, in IB-A group, the BaxlBcl-2 ratio significantly increased when compared with H group, and the result shows that apoptosis was induced, which is accordance with the Ca2+ overloading found in the present study. But those all recover to the Pre-H level and the found is consistent with the ratio of mRNA expression level, which demonstrated that the ability of anti-apoptosis has enhanced. |
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