The Apoptosis, Degradation Effects And Possible Mechanisms Of ALLN, UPFâ…¡ Alone And Combination Applying In Rat Skeletal Muscle Cells Of Serum Starvation

Objective：To study the apoptosis, degradation effects and possible mechanisms byusing calpain inhibitor ALLN (N-acetyl-Leu-Leu-Norleucinal) and Ubiquitin-Proteasome Fraction Ⅱ(UPFⅡ)alone and their combination in primacy cultural SDrat skeletal muscle cells of serum starvation.Methods：The skeletal muscle cells of SD rat,3-5days old, were cultivated in vitroand identified by means of HE staining and skeletal muscle actin (α-sarcometric actin)immunofluorescence. The skeletal muscle cells were timely observed, picture-takenand recorded under inverted microscope during the process of above. When theskeletal muscle cells were mature and stable after five or six generations of spreading,they were randomly divided into5groups:(1) Complete medium: group A (blankcontrol group);(2) Basal medium: group B（DMEM negative control group), groupC (DMEM+UPFⅡ), group D (DMEM+UPFⅡ+ALLN), group E (DMEM+ALLN). The growth vigor of skeletal muscle cells constituting was determined byMTT assay and each group’s Calpain-3, MuRF-1and Atrogin-1in the expression ofmRNA level were detected by Real-time fluorescent quantitative PCR.Results：1.The results, determined by MTT, of group B and group D skeletal muscle cellsurvival ratio showed similarity decrease (57.89%±5.07%),(56.94±6.02) respectively(P>0.05)and group C decreased significantly (41.22%±4.87%) compared with groupA (P <0.05). However, compared with the previous three groups (B,C,D), group Eincreased significantly (69.72%±6.25%)(P <0.05);2.Real-time fluorescent quantitative PCR results showed that: group(BCDE)’sCalpain-3mRNA, MuRF-1mRNA, Atrogin-1mRNA expression quantity increased and expressed respectively23.71±0.10/23.18±0.08/22.22±0.10times,24.52±0.07/24.10±0.07/23.29±0.05times,23.78±0.07/23.11±0.05/22.15±0.07times,22.60±0.04/22.37±0.10/21.65±0.08times compared withthe blank control group A, obviously different (P <0.05); Each gene expression ofgroup E was significantly lower than group(BCD)(P <0.05) and each gene expressionof group D was significantly lower than group C (P <0.05); There was no significantdifferences between group B and group D (P>0.05).Conclusion：A moderate amount of calpain inhibitor ALLN can effectively inhibitthe auxo-action of exogenous ubiquitin-proteasome (UPFⅡ)to the apoptosis anddegradation of skeletal muscle cells; The ubiquitin/protease system may be subject tothe pilot control of the calpain system during the process of the apoptosis anddegradation of skeletal muscle cells.