The Effects Of Wnt/β-catenin Signalling On Proliferation&differentiation Of Porcine Satellite Cells And The Preliminary Research Of The Mechanism

Skeletal muscle tissue is composed by the terminally differentiated plurinuclearmuscle fiber, the number of muscle fibers is essentially the same in postnatalmammals. When suffered from external stimuli (injury, exercise, etc.), silent skeletalmuscle satellite cells will be activated, isolating from the muscle fiber sarcolemmaand basement membrane (anatomical position), and then proliferate and directionallydifferentiate, causing the muscle fiber hypertrophy. Many studies have shown that theWnt/β-catenin signaling pathway plays an important role in the mammaliandevelopment, and has impacts on embryonic development, cell proliferation,differentiation, and apoptosis. The role of this signaling pathway in the generation ofskeletal muscle have been demonstrated. Wnt is necessary to skeletal muscleformation in Xenopus laevis, poultry and mammals, which determines the satellite cellfate and the key regulatory factors for myoblast terminal differentiation. Studies haveshown that epigallocatechin-3-gallate (EGCG), not only inhibits the proliferation ofcells of many types. In addition, EGCG has an inhibitory effect on Wnt/β-cateninsignaling pathway in vivo and in vitro.As an important meat-producing animal and model animal, pig is of great valuein production and scientific researchs. Therefore, we chose pig as the subject in thisstudy, dissociated and cultured porcine skeletal muscle satellite cells in vitro,investigated the effects of Wnt/β-catenin inhibitior EGCG on the porcine skeletalmuscle satellite cell proliferation and differentiation, analysed changes of cell cycleand detected the impact of the beta-associated protein on the expressions of markergenes during skeletal muscle satellite cell proliferation and differentiation. Theobjective is to preliminaryly study the the mechanisms of Wnt/β-catenin signalingpathway in the porcine skeletal muscle satellite cell proliferation and differentiationand provide a theoretical basis for skeletal muscle satellite cells in muscleregeneration and fiber hypertrophy.1,The effect of EGCG on porcine skeletal muscle satellite cell proliferationThe Wnt/β-catenin signaling pathway inhibitor EGCG was used to treat skeletal muscle satellite cells. Then the proliferation were analyzed with MTT, and Westernblotting. The results showed that EGCG inhibit porcine skeletal muscle satellite cellproliferation in a time-and dose-dependent manner. These results indicate that:EGCG inhibited the proliferation of porcine skeletal muscle satellite cells as well asthe expression of β-catenin.2,The effect of EGCG on porcine skeletal muscle satellite cell differentiationThe Wnt/β-catenin signaling pathway inhibitor EGCG was used to treat skeletalmuscle satellite cells. Then the differentiation were analyzed withimmunofluorescence and Western blotting. Compared to the control, under theinverted microscope, we observed EGCG promote the differentiation of musclesatellite cells; Western blot analysis showed that the quantity of MyoG and MyHCprotein rised; more MyoG and MyHC generation was also found byimmunofluorescence staining. Furthermore,EGCG increase the Fusion Index ofmuscle satellite cells that on differentiatial conditions. These results suggested thatEGCG enhanced the ability of the muscle satellite cell differentiation and theformation of myotubes.3,The effect of EGCG on cell cycle of porcine skeletal muscle and the Wnt/β-catenin signaling pathway and the regulatory factors during skeletal muscleproliferation and differentiationFlow cytometry analyses of cell cycle revealed that the percentage of cells in G1phase was increased but G2and S phases decreased after EGCG treatment. Thissuggested that the cells were blocked in the G1phase and cell proliferation wasinhibited.The Wnt/β-catenin signaling pathway inhibitor EGCG was used to treatskeletal muscle satellite cells. Immunofluorescence staining was used to detected theβ-catenin subcellular localization during cell proliferation and differentiation, as wellas the protein expression of cyclin D1, which are Wnt/β-catenin downstream targetgenes. Compared with the control group, EGCG reduced the amount of β-cateninprotein in porcine skeletal muscle satellite cell either during proliferation ordifferentiation, so that nuclearβ-catenin significantly reduced.These results indicated that, EGCG inhibited porcine skeletal muscle satellite cellproliferation by wnt/β-catenin signaling pathway and Cyclin D1were inhibited.EGCG promoted skeletal muscle satellite cell differentiation.