Background/Aims: Skeletal muscle ischemia/reperfusion (I/R) injury is a common and severe disease. Sonic hedgehog (Shh) plays a critical role in post-natal skeletal muscle regeneration. In the present study, the role of Shh in skeletal muscle I/R injury and the mechanisms involved were investigated. Methods: The expression of Shh, AKT/mTOR/p70S6K and apoptosis pathway components were evaluated following tourniquet-induced skeletal muscle I/R injury. Then, mice were subjected to systemic

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... ration of cyclopamine or one-shot treatment of a plasmid encoding the human Shh gene (phShh) to examine the effects of Shh on I/R injury. Moreover, mice were subjected to systemic administration of NVP-BEZ235 to investigate the role of the AKT/mTOR/p70S6K pathway in Shh-triggered skeletal muscle protection. Results: We found that the levels of Shh, AKT/mTOR/p70S6K pathway components and Cleaved Caspase 3 and the Bax/Bcl2 ratio initially increased and then decreased at different time points post-I/R injury. Moreover, Shh protected skeletal muscle against I/R injury by alleviating muscle destruction, reducing interstitial fibrosis and inhibiting apoptosis, and these protective effects were abrogated when the AKT/mTOR/p70S6K pathway was inhibited. Conclusion: Collectively, these data suggest that Shh signaling exerts a protective role through the AKT/ mTOR/p70S6K signaling pathway during skeletal muscle I/R injury. Thus, Shh signaling may be a therapeutic target for protecting skeletal muscle from I/R injury. Fig. 3. Effects of phShh treatment on skeletal muscle destruction and fibrosis in response to I/R injury. (A) Skeletal muscle was treated with phShh after ischemic injury, and immunofluorescence staining for Shh (green) was performed on cross sections of phShh-treated and control vector-treated skeletal muscle 7 days after I/R injury; nuclei were labeled with DAPI (blue). Scale bars, 50 μm. n=5/group. (B) Western blotting was performed to estimate the protein expression of Shh, Gli1 and Gli2 in phShh-treated, control vector-treated and untreated skeletal muscle 7 days after I/R injury. Muscle from the uninjured contralateral limb served as the control group. n=3/group. (C) H&E staining and Masson's trichrome staining were performed on muscle sections from the phShh treatment group and the control vector treatment group 7 days after I/R injury (C left, H&E staining, magnification = ×400; C right, Masson's trichrome staining, magnification = ×400. Scale bars, 50 μm). n=3/group. (D) Destruction scores determined by H&E staining of skeletal muscle were used to estimate the degree of muscle damage. (F) The collagen area, based on Masson's trichrome staining, was used to evaluate the fibrotic tissue and was determined by quantification of the blue staining area. Fiji ImageJ software was used to quantify the percentage of collagen area. All data are presented as the mean ± SEM; * indicates a statistically significant difference between the phShh treatment group and the control vector treatment group (p < 0.05). Figure. 3. Zeng et al.: Shh Protects Skeletal Muscle Against I/R Injury decreased (Fig. 4A and B) . The results indicated that the AKT/mTOR/p70S6K signaling pathway was activated in skeletal muscle in the early recovery phase of I/R injury. Fig. 7. phShh treatment exerted an anti-apoptotic effect following skeletal muscle I/R injury. (A) The time- course expression of Cleaved Caspase 3, Bax and Bcl2 after 3 hours of unilateral hindlimb ischemia and 1, 3, 5, 7, or 14 days of reperfusion were estimated with western blotting. n=3/group. * indicates a statistically significant difference between each I/R group and the control group (p < 0.05). (B) phShh treatment was used to over-express Shh, and/or cyclopamine treatment was used to inhibit the Shh pathway. Then, the expression of Cleaved Caspase 3 protein, and the ratio of Bax/Bcl2 in skeletal muscle 7 days after I/R injury were estimated by western blotting. n=3/group. # indicates a statistically significant difference between the I/R+phShh group and the I/R+control vector group, * indicates a statistically significant difference between each I/R group and the I/R+phShh+cyclopamine group (p < 0.05). (C) TUNEL staining was used to examine the apoptosis of muscle cells in the phShh-treated and control vector-treated skeletal muscle 7 days after I/R injury. In addition, the apoptotic index was used to quantify the level of apoptosis. n=3/group. (D) phShh treatment was used to over-express Shh, and/or NVP-BEZ235 treatment was used to inhibit the AKT/mTOR/p70S6K pathway. Then, the expression of cleaved caspase-3 protein and the ratio of Bax/ Bcl2 in skeletal muscle 7 days after I/R injury were estimated by western blotting. n=3/group. * indicates a statistically significant difference between each I/R group and the I/R+phShh+NVP-BEZ235 group (p<0.05). All data are presented as the mean ± SEM.