| Background:The nuclear transcription factor NF-?B participates in the regulation of genes expressions related to apoptosis, inflammation, cancer, oxidative stress and autoimmunity. According to the previous reports, NF-?B can regulate the transcriptional level of ubiquitin ligase MuRF1 that is a necessary factor resulting in disuse muscle atrophy via activating the NF-?B signaling pathway. Therefore, it suggests that the NF-?B signaling pathway plays an important role in the pathogenesis of disuse muscle atrophy. Hibernators have a novel protection mechanism for inhibiting disuse induced muscle atrophy during the long hibernation. Therefore, more and more scholars attempt to find preventive measures of disuse muscle atrophy by studying hibernators.Objective:To explore the regulation of NF-?B to target genes downstream MuRF1 in hibernation process by study the changes of mRNA and protein expression level of NF-?B signaling pathway and MuRF1 in soleus and extensor digitorum longus in ground squirrels for different period of hibernation, further to studied the possible mechanisms of anti-muscle atrophy of ground squirrels (Spermophilus dauricus).Methods:1. The relative expression amount of MuRF1 mRNA, NF-?B family member IKK? mRNA and p50 mRNA of soleus muscle and extensor digitorum longus for different period of hibernation were tested by real-time fluorescent quantitative PCR technique.2. The relative protein expression amount of MuRF1, NF-?B family member IKK? and p50 of soleus muscle and extensor digitorum longus for different period of hibernation were tested by Western Blot technique.Results:(1) The ratio of muscle and body weightThe ratio of muscle and body weight in soleus:Compared with Preceded Hibernation group, the changes of the ratio of muscle and body weight of soleus muscle in Hibernation group?Interbout arousal group and Posterior Hibernation group were not statistically significant(P>0.05). While the muscle weight of soleus muscle in Posterior Hibernation group decreased by 17.2%(P<0.05) comparing with Preceded Hibernation group, and a little decrease in Hibernation group and Interbout arousal group.The ratio of muscle and body weight in extensor digitorum longus:Compared with Preceded Hibernation group, the muscle weight of extensor digitorum longus decreased by 16.8%(P<0.05),13.9%(P<0.05) and 18.2%(P<0.05) in Hibernation group, Interbout arousal group and Posterior Hibernation group, respectively. However the changes of the ratio of muscle and body weight of extensor digitorum longus in Hibernation group? Interbout arousal group and Posterior Hibernation group were not statistically significant(P>0.05).(2) Changes of expression of IKK? mRNASoleus:Compared with Preceded Hibernation group, the expression of IKK? mRNA in Interbout arousal group increased by 49.4%(P<0.05), while decreased by 74.22% (P<0.05) in Posterior Hibernation group. Compared with in Hibernation group, the expression of IKK? mRNA in Posterior Hibernation group decreased by 80.7%(P<0.01). Compared with Interbout arousal group, the expression of IKK? mRNA in Posterior Hibernation group decreased by 82.7%(P<0.01)extensor digitorum longus:Compared with Preceded Hibernation group, the expression of IKK? mRNA in Hibernation group and Interbout arousal group increased by 73.6%(P<0.05) and 158.4%(P<0.01) respectively, while decreased by 90.8%(P<0.01) in Posterior Hibernation group. Compared with in Hibernation group, the expression of IKK? mRNA in Interbout arousal group increased by 48.9%(P<0.01), while decreased by 94.7%(P<0.01) in Posterior Hibernation group. Compared with Interbout arousal group, the expression of IKK? mRNA decreased by 96.4%(P<0.01) in Posterior Hibernation group.(3) Changes of expression of p50 mRNA and proteinThe expression of mRNA in soleus:Compared with Preceded Hibernation group, the expression of p50 mRNA in Interbout arousal group increased by 37.1%(P<0.05).There are no significant changes in other groups (P>0.05).The expression of protein in soleus:Compared with Preceded Hibernation group, the expression of p50 protein in Hibernation group had a little increase, but no significance, while increased by 20%(P<0.01) and 39.2%(P<0.01) in Interbout arousal group and Posterior Hibernation group, respectively. Compared with in Hibernation group, the expression of p50 protein in Posterior Hibernation group increased by 27.2%(P<0.01) Compared with Interbout arousal group, the expression of p50 protein in Posterior Hibernation group increased by 16%(P<0.05)The expression of mRNA in extensor digitorum longus:Compared with Preceded Hibernation group, the expression of p50 mRNA in Interbout arousal group increased by 43.4%(P<0.05), while decreased by 47.2%(P<0.05) in Posterior Hibernation group. There are no significant changes in other groups (P>0.05). Compared with in Hibernation group, the expression of p50 mRNA in Posterior Hibernation group decreased by 56.1% (P<0.01).Compared with Interbout arousal group, the expression of p50 mRNA in Posterior Hibernation group decreased by 63.2%(P<0.01)The expression of protein in extensor digitorum longus:Compared with Preceded Hibernation group, the expression of p50 protein in group increased by 19.1%(P<0.05)and 37.6%(P<0.01) in Hibernation group and Posterior Hibernation group, respectively, while decreased by 14.7%(P<0.05) in Interbout arousal group. Compared with in Hibernation group, the expression of p50 protein in Interbout arousal group decreased by 28.34% (P<0.01), while increased by 15.5%(P<0.05) in Posterior Hibernation group. Compared with Interbout arousal group, the expression of p50 protein in Posterior Hibernation group increased by 61.3%(P<0.01)(4) Changes of expression of MuRF1 mRNA and proteinThe expression of mRNA in soleus:Compared with Preceded Hibernation group, the expression of MuRF1 mRNA in Interbout arousal group increased by 3.4 times (P<0.01) Compared with in Hibernation group, the expression of MuRF1 mRNA in Interbout arousal group increased by 1.4 times(P<0.01). Compared with Interbout arousal group, the expression of MuRF1 mRNA in Posterior Hibernation group decreased by 63.8% (P<0.01)The expression of mRNA in extensor digitorum longus:Compared with Preceded Hibernation group, there is a little increase in the expression of MuRf1 mRNA in Hibernation group and Posterior Hibernation group, and a little decrease in Interbout arousal group, but have no significance (P>0.05). There are no significant changes in other groups (P>0.05).The expression of protein in extensor digitorum longus:Compared with Preceded Hibernation group, the expression of MuRF1 protein in group increased by 87%(P<0.01) and 1.1 times (P<0.01) in Hibernation group and Interbout arousal group, respectively. Compared with in Hibernation group, the expression of MuRF1 protein in Posterior Hibernation group decreased by 45.4%(P<0.01). Compared with Interbout arousal group, the expression of MuRFl protein in Posterior Hibernation group decreased by 51.7% (P<0.01).The expression of protein in soleus:Compared with Preceded Hibernation group, the expression of MuRF1 protein in group increased by 1.8 times (P<0.01) in Hibernation group. Compared with in Hibernation group, the expression of MuRF1 protein in Interbout arousal group decreased by 61.1%(P<0.01), the expression in Posterior Hibernation group decreased by 54.2%(P<0.01)Conclusions:1. The muscle weight of ground squirrels is reduced during hibernation, and the mass of fast muscle (extensor digitorum longus) and slow muscle (soleus) muscle are significantly reduced after hibernation. It is indicated that skeletal muscle is atrophied, but the ratio of muscle and body weight have no significant changes. The normal exercise capacity of skeletal muscle was not affected by the decrease of muscle weight.2. The expression of IKK?, p50 and MuRFl all increase during the hibernation process of ground squirrels, especially the MuRF1 protein expression of extensor digitorum longus in Hibernation group have a significant increment. Increasing each of those three genes will induce the disuse muscle atrophy for non-hibernators, and skeletal muscle protein degradation pathways will be rising with the increment of those three genes, which is the primary cause of skeletal muscle atrophy for ground squirrels.3. During the hibernation of ground squirrels, MuRF1 gene expression of slow muscle will increase in Hibernation group and Interbout arousal group but decrease in post-hibernation, and gene expression of slow muscle has no significant changes. It suggests that the regulation of MuRF1 expression is different for fast and slow muscle in hibernation, and another signal pathway participates in the regulation of the related genes expressions.4. The protein expression changes of p50 coincides with the genes expression changes of MuRFl during hibernation of ground squirrels, which suggests hibernation-period p50 can regulate the expression of MuRFl. However, their expression will change when post-hibernation perhaps because another signal pathway participates in the regulation of MuRF1 expressions and make the protein degradation pathways recovery. Ground squirrels can inhibit the expression of IKK?because the expression of IKK? is increased in Hibernation group and Interbout arousal group (especially in Interbout arousal group) when Ground squirrels hibernating, however, which will be recovered after post-hibernation. |
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