The Effect And Mechanism Of HGF Regulating TGF-β1-Induced Fibrotic Transformation Of Skeletal Muscle Fiber

Objectives:Explore an effective，mature method to isolate and culture skeletal muscle fibers invitro, and then observe the role of TGF-β1-induced fibrotic transformation of skeletalmuscle fibers. Find the effect and mechanism of HGF regulating TGF-β1-induced fibrotictransformation of skeletal muscle fibers.These results may provide a new strategy andtheoretical basis for clinical treatment of fibrosis in denervated laryngeal muscle.Methods:1. Isolate and culture skeletal muscle fibersThe skeletal muscle was digested by collagenase type II perfusion through theabdominal aorta in the experimental group and cut from the right and left extensordigitorum longus(EDL) of5-week-old male C57BL/mice.Then the muscle was rinsedin D-Hanks solution and incubated in a shaker tube containing collagenase type II andtype I.In the control group the skeletal muscle directly removed after cervicaldislocation.After digested by collagenase type I, use a wide-bore pipette gently triturateeach muscle belly throughly separated.After a while, plate the intact, living, singlemuscle fibers and compare the quantites of fibers and survival in two groups.2. TGF-β1induces CTGF and α-SMA expression in skeletal muscle fibers andmyotubesC2C12cells were cultured in DMEM supplement with10%fetal bovine serum(FBS).Differerntiation of myoblasts into myotubes was achieved by growing the cells toconfluence and changing the serum supplementation from10%FBS to2%horse serum(differerntiation medium). After72hours of exposure to differerntiation medium, cellswere cultured in serum-free DMEM containing different concentrations of TGF-β1(0ng/ml,0.1ng/ml,1ng/ml,5.0ng/ml).After12hours incubation,the transcript and proteinlevels of CTGF and α-SMA were assayed by realtime PCR and Western-Blot.Single muscle fibers were cultured in DMEM supplement with20%fetal bovineserum,10%horse serum and1%Chicken embryos extract(CEE).Single fibers wererandomly divided into control group and experimental group.The medium of experimentalgroup was supplemented with0.5ng/ml of TGF-β1. After12hours incubation, thetranscript and protein levels of CTGF and α-SMA were assayed by realtime PCR and Western-Blot.3. HGF depress the production of CTGF and α-SMA induced by TGF-β1in SkeletalMuscle fiberSingle fibers were randomly divided into control group，TGF-β1group(0.5ng/mlTGF-β1) and HGF group(0.5ng/ml TGF-β1+4ng/ml HGF). After12hours incubation,thetranscript and protein levels of CTGF and α-SMA were assayed by realtime PCR andWestern-Blot.The location and relative abundance of CTGF andα-SMA were detected byimmunofluorescence.Results:1.Comparison of quantites and survival of fibers in two methodsStatistics of the quantites of the fibers at╳5magnification were compared.The numberof fibers which were obtained by collagenase type II perfusion was （32.17士2.09）ineach field of vision. The number of fibers that gained from triturated muscle belly was（10.58士1.15）in each field of vision. The difference was statistically significant (P<0.05).And it also has prolonged survival in24hours,48hours and72hours. Thesurvival differences were statistically significant (P <0.05).But the operating time andprocess of this method are relatively complex,requiring long-term repeated practice andexploration.2. Comparison of different concentrations of TGF-β1induced CTGF and α-SMAexpression in myotubesCTGF mRNA expression of myotubes in TGF-β1groups compared with blankcontrol group were significantly higher. The expression of1ng/ml group was the highest,16.03times higher than blank control group.The difference was statistically significant (P<0.05). α-SMA mRNA expression of myotubes in TGF-β1groups compared with blankcontrol group were significantly higher. The expression of1ng/ml group was the highest,4.52times higher than blank control group.The difference was statistically significant (P<0.05). CTGF and α-SMA protein expression of myotubes in TGF-β1groups comparedwith blank control group were both significantly higher. The CTGF protein expression of1ng/ml group was the highest,1.88times higher than blank control group. The α-SMAprotein expression of1ng/ml group was the highest,1.59times higher than blank controlgroup.The differences were statistically significant (P <0.05).3. The role of TGF-β1induced CTGF and α-SMA expression on single fibersCompared with blank group, TGF-β1can promote skeletal muscle fibers CTGF and α-SMA expression at mRNA level. TGF-β1induced CTGF mRNA expression ofskeletal muscle cells9.55times than the blank control group. TGF-β1inducedα-SMAmRNA expression of skeletal muscle cells26.82times than the blank control group.statistical significant(P<0.05).Weston blot results also showed that TGF-β1can promoteCTGF and α-SMA protein expression. CTGF protein expression was3.63times than thecontrol group,and α-SMA protein expression was3.38times than the control group.These differences were statistically significant (P <0.05).4. HGF depress the production of CTGF and α-SMA induced by TGF-β1in single fibersCompared with TGF-β1group, HGF can depress skeletal muscle fibers CTGF andα-SMA expression at mRNA and protein level. CTGFmRNA expression of TGF-β1group was6.57times higher than HGF group. α-SMA mRNA expression of TGF-β1group was2.64times higher than HGF group. CTGF protein expression of TGF-β1groupwas2.2tim1es higher than HGF group. α-SMA protein expression of TGF-β1group was1.49times higher than HGF group. These differences were statistically significant (P<0.05). immunofluorescence all detected positive signals. The signal of HGF group isweaker than that of TGF-β1group.Conclusion:1. The method of collagenase perfusion has more fibers, prolonged survival than theother tranditional method.But operating time and process of this method arerelativelycomplex,require long-term repeated practice and exploration.2. TGF-β1can promote skeletal muscle fibers and myotubes fibrosis related proteinCTGF and α-SMA expression.It plays an important role in the process of skeletal musclefibrosis.3. HGF can depress TGF-β1induced fibrosis in skeletal muscle.This cytokine provides anew idea of treatment for denervated laryngeal muscle fibrosis at the cellular andmolecular level.