Effects Of Salidroside On Weightlessness-induced Muscle Atrophy And Duchenne Muscular Dystrophy As Well As Their

First: Effects of Salidroside on the formation of weightlessness-induced muscleatrophy and their mechanismsIt is known that during the spaceflight of the astronauts, especially the prolongedspaceflight, weightlessness can cause skeletal muscle atrophy as well as the mostserious atrophy in anti-gravity muscles such as the soleus muscle, which ischaracterized by the loss of muscle mass, decrease of muscle fiber cross-sectionalarea, and the slow-to-fast-twitch myofiber type transition. However, the existingcountermeasures can not ease the muscle atrophy, which could seriously affect theastronauts’ performance of missions and their healthy recovery after returning to theground. In order to meet the needs of building manned lunar and long-terminterplanetary missions of our country in near future, new effective countermeasures,including drugs, should be developed.Our preliminary research indicated that TGF-β/Smad3pathway is activated inhindlimb unloading (HU) induced soleus muscle atrophy. Moreover, Smad3deficientmice are protected from unloading-induced loss of soleus muscle mass, muscle fiberatrophy and slow-to-fast fiber type transition, which suggested that Smad3may be anattractive target for identifying the effective drugs in the treatment of themicrogravity-induced muscle atrophy. Since the natural plant Rhodiola and itseffective active ingredient Salidroside can inhibit the expression of TGF-β1in kidneyor liver tissues, we speculate that Rhodiola/Salidroside could reduce the formation ofmuscle atrophy by inhibiting the activation of TGF-β/Smad3signaling pathway whichis induced by HU. In the present study, wild-type C57BL/6mice, which were gavageddaily with Salidroside or an equivalent amount of PBS for28days, were subjected to14days of HU to induce skeletal muscle atrophy. After that, we explored the role ofp-Smad3in the effect of salidroside on weightlessness-induced muscle atrophy and itsregulatory mechanism, through comparative analysis of muscle weight loss, fibercross-sectional area, composition of different muscle fibers and expression ofTGF-β1/Smad3signaling pathway. The main research results are as follows: 1. After14days of HU, the weight and fiber cross-sectional area of hindlimb muscles,especially slow-twitch soleus muscles, were significantly decreased in PBS treatedmice, which were inhibited in Salidroside treatment mice. It suggested thatSalidroside could protect the soleus muscle and its fibers from HU-induced atrophy.2. Skeletal muscle-specific ubiquitin ligases, Atrogin-1and MuRF1, weredramatically upregulated in the soleus muscles of PBS-treated mice with HU-inducedmuscle atrophy. However, this upregulation was significantly reduced inSalidroside-treated muscles, suggesting that Salidroside could inhibit HU-inducedmuscle atrophy by blocking the activation of the ubiquitin-proteasome pathway.3. In PBS treated mice with HU for14days, the content of slow twitch fibers (MHC-Iand MHC-IIa) decreased but the content of fast-twitch fibers (MHC-IIb) increased insoleus muscles, while there was no changes in the content of MHC-IIa in Salidrosidetreated mice after14days of HU, and also the content of MHC-IIb is significantlylower in Salidroside compared to the PBS treated mice., This result indicated thatSalidroside has a dramatic resistance of slow-to-fast fiber type transition in soleusmuscles by inhibiting HU-induced decrease of MHC-IIa and the HU-induced increaseof MHC-IIb.4. Western blot results showed that salidroside inhibited the HU-induced decrease ofTroponin I-SS and increase of Troponin I-FS, which represented the content of slowor fast muscle fibers respectively. Therefore, this result further validated the inhibitoryeffect of Salidroside on HU-induced slow-to-fast fiber type transformation.5. TGF-β1/Smad3signaling pathway was activated after14days of HU. Salidrosidetreatment not only reduced the expression of TGF-β1/Smad3signaling pathway in thesoleus muscles of ground-based control mice, but also significantly inhibitedHU-induced activation of this pathway.6. pGL-3-Atrogin-1was successfully constructed by ligating the promoter region ofthe Atrogin-1gene with the luciferase reporter gene in the pGL3-Basic vector, andthen co-transfected with Smad3expression plasmid into skeletal muscle cells. Wefound that the overexpression of Smad3can enhance the activity of Atrogin-1promoter, which suggested that p-Smad3could activate the transcription of Atrogin-1.7. We discovered that there is a putative binding site for Smad3in the upstream ofAtrogin-1gene (-2115to-2107) using bioinformatic comparison. However, ChIPresults showed p-Smad3failed to directly bind to this site in unloaded soleus muscles,suggesting that HU-induced p-Smad3activation indirectly rather than directly regulates the expression of Atrogin-1through interaction with other proteins.8. Among the proteins which interact with Smad3, FOXO is the only one that can playa direct regulation effect on the transcription of Atrogin-1. ChIP-re-ChIP resultshowed that a FOXO binding site can be detected in Smad3antibody precipitatedDNA fragments sonicated from unloaded soleus muscles. The results showed thatp-Smad3indirectly influences the transcription of Atrogin-1by binding with FOXO inHU-induced soleus muscle atrophy.9. In addition, ChIP-re-ChIP results demonstrated that Salidroside treatment decreasedthe expression of Atrogin-1by reducing binding enrichment of the p-Smad3andFOXO on Atrogin-1promoter region.10. In order to verify if p-Smad3mediates the inhibitory effect of Salidroside onHU-induced slow to fast-twitch muscle fiber type transformation, we applied Smad3knockout mice (Smad3+/-) and their littermate wild-type mice (Smad3+/+) to HU.The result confirmed that Smad3knockout mice can also fight against HU-inducedMHC-IIa reduction and increased MHC-IIb. It suggested that salidroside may inhibitthe HU-induced myofiber type transition by blocking the HU-induced p-Smad3activation.11. Reporter gene constructs carrying MHC-IIa or MHC-IIb promoter sequence weresuccessfully constructed. The luciferase activity of MHC-IIa and MHC-IIb can bedecreased or increased respectively by TGF-β1alone, and this effect would be moresignificant with the overexpression of Smad3. It suggested that p-Smad3can inhibitthe expression of MHC-IIa but increase the expression of MHC-IIb.12. Comparing the upstream regions of the transcription start site in rats and micewith bioinformatics analysis, it indicates that there are three or two conservedSmad-binding elements (SBEs) in the promoter regions of MHC-IIa or MHC-IIb generespectively.13. ChIP results confirmed that p-Smad3can directly bind to SBEs which are close tothe transcription start sites and these SBEs located at-54~-44bp upstream of MHC-IIagene and-630~-622bp upstream of MHC-IIb gene separately.14. pGL-3-MHC-IIa and pGL-3-MHC-IIb with mutated Smad-binding sites weresuccessfully generated by site-directed mutation strategy. Then the inhibition ofMHC-IIa or the enhancement of MHC-IIb were reversed by p-Smad3, confirming theSBE sites are indispensable in the regulation on the expression of MHC-IIa andMHC-IIb of p-Smad3. 15. ChIP experiments showed that Salidroside treatment reduced the bindingenrichment of the p-Smad3onto the promoter regions of MHC-IIa and MHC-IIbgenes by inhibiting the expression of HU-induced p-Smad3, thus combating theHU-induced decrease of MHC-IIa and increase of MHC-IIb.In summary, we demonstrated that Salidroside could inhibit the simulatedweightlessness-induced antigravity soleus muscle atrophy, and the transformationfrom slow fiber to fast fiber. And also we revealed the regulation of Smad3in theeffect of Salidroside against weightlessness-induced muscle atrophy. This study notonly provides a potential effective molecular target for protection against muscleatrophy, but also shows a promising future in the treatment of of muscle atrophy withRhodiola and its main pharmacologically active ingredient Salidroside.Second: Study of Salidroside in treating animals with Duchenne musculardystrophyDuchenne muscular dystrophy (DMD) is a clinically severe fatal disease whichis caused by deletions in dystrophin gene located on human X chromosome. DMDpatients have no expression of dystrophin in their muscle cells, so the musclecontraction can cause repeated membrane damages of muscle cells, continuousinflammatory response and fibrosis. The high expression of TGF-β1is believed toplay a critical role in the formation of muscle fibrosis. Bsed on our previous researchthat Salidroside can inhibit the expression of TGF-β1and HU can reduce thecontraction of hindlimb muscle, we treated the mdx mice, a well-recognized model ofDMD disease, with Salidroside as well as immobilization. The main findings are asfollows:1. HE staining result showed that skeletal muscles of mdx mice had severeinflammatory response. Salidroside significantly reduced the inflammatory responsein mdx muscles and rescued the expression of inflammatory cytokines to normallevels of wild-type mice.2. Compared to wild-type mice, the expression of myogenic differentiation factorssignificantly increased in mdx muscles. Salidroside treatment inhibited the expressionof myogenic differentiation factors, suggesting the reduced repair-regenerativeresponse in Salidroside treated mdx mice.3. Compared to wild-type mice, the formation of fibrosis and expression of fibroblastgrowth factors in the muscles of mdx mice increased significantly, which has the most significant increase in collagen Iα1. Salidroside significantly inhibited the expressionof collagen Iα1and the formation of muscle fibrosis in mdx mice.4. Compared to wild-type mice, the expression of MHC-I and MHC-IIa genes weresignificantly reduced while the expression of MHC-IId and MHC-IIb were increasedin mdx muscles. Salidroside treatment significantly reduced the expression ofMHC-IId and MHC-IIb, suggesting that Salidroside could inhibit the slow-to-fastmyofiber type transiton in mdx mice.5. TGF-β1/Smad3signaling pathway was activated in mdx muscles, but Salidrosidetreatment significantly inhibited the activity of TGF-β1/Smad3signaling pathway inmdx muscles, suggesting that Salidroside could ameliorate pathological damages ofskeletal muscles of mdx mice by inhibiting the activation of TGF-β1/Smad3signalingpathway.6. Hindlimb skeletal muscles of mdx mice, especially the gastrocnemius muscle,showed an obvious inflammatory response. However,14days of HU significantlyreduced inflammatory response, centrally located nucleic number and necrotic musclefibers in mdx gastrocnemius muscles.7. Muscle fiber atrophy occurred in mdx muscles during HU, which implied that bymeans of HU to reduce pathological damage of mdx mouse skeletal muscles, muchattention should be paid for the protection against the formation of muscle atrophy.These results suggest that salidroside could alleviate pathological damages ofDMD patients by inhibiting TGF-β1/Smad3signaling pathway and fibrosis.Immobilization caused by hindlimb unloading can reduce the inflammatory andnecrotic response in mdx mice. This study provides a reference for the comprehensivetreatment of DMD disease. Whether the combination of immobilization andsalidroside can reduce the pathological damage and other negative effects of DMD,such as muscle atrophy, still remains to be further studied.