Effects Of Moo On Tgf-β2 Signal Transduction When The Differentiation Of Myoblasts Into Myocardial-like Cells

Background and Objective:Recently the research on cellular transplantation for myocardial infarction which can rebuilt cardiac muscle cell and retrieve the function of the necrosed myocardium was excellent cure method.transplanting skeletal muscle myoblast,which is precursor cell of musculature and posses potentiality of differentiating to skeletal muscle or cardiac muscle.to the myocardium to replace the damaged myocardium and scar tissue developed greatly.Our research group is investigating the protective effect of Chinese herbal medicine Morinda Officinalis How Oligosaccharide(MOO) on rat hearts.Recent research initially confirmed that Morinda Officinalis How Oligosaccharid can obviously promote the proliferation of myoblasts.Morinda Officinalis How Oligosaccharid can increase the expression of myoblasts' transforming growth factor-betal(TGF-β1) to an extent.However,there are no reports on effects of Morinda Officinalis How Oligosaccharid on TGF-β2 signaling transduction so far. Therefore,on the basis of previous study,we use primary cell culture, immunocytochemistry and western blotting to explore effects of Morinda Officinalis How Oligosaccharid on TGF-β2 signal transduction when the differentiation of myoblasts into myocardial-like cells.Materials and Methods: The skeletal muscles were isolated from the limbs of the neonatal SD rats(1～3 days after birth) and minced in phosphate buffer solution.They were dissociated with 0.05%collagenaseⅡand 0.125%or 0.25 pancreatin.After dispersed,the cells were collected,purified and incubated in 100ml culture bottles or 6 pore plates(the cell density was adjusted to 3×10~5 cells/ml with DMEM/F-12 culture liquid).Cultured myoblasts were divided into six groups:blank group;5-Aza-dc group(4μmol/ml);high dose group(500μg/ml);medium dose group(300μg/ml);low dose group(100μg/ml); medium dose group combined 5-Aza-dc group(MOO300μg/ml+5-Aza-dc 4μmol/ml).The following indices were detected:myoblast morphology;the expression of Desmin in immunocytochemistry;the expression of TGF-β2 and Smad4 in immunocytochemistry;the expression of phosphorylated Smad2/3(p-Smad2/3) and Smad7 in western blotting.Data were analyzed by the SPSS11.0 statistical software package.Results were expressed as mean±standard deviation((?)±s).Different groups were compared through one-way ANOVA,followed by LSD.P-values of less than 0.05 was considered significant.Results:1.Myoblast morphologyNormal myoblasts cultured for 24 to 48 hours were in fusiform with high refractive index observed by inverted microscope.Cells grew intensively and proliferated obviously for 72 hours,increased fusiform cells became slender and multinuclear primary Tube-form myotubes for 4 to 5 days,Tube-form myotubes confluened with each other after 6 days.With time,confluened Tube-form myotubes increased and became nets.Spontaneous impulse was seen in well differentiated myotube after 5 days.Soon rhythmic contraction showed up.Impulse frequence was effected obviously by temperature,the lower temperature was and the slower Impulse was.2.Determination of Desmin in immunocytochemistryDesmin was detected by immunocytochemistry and the results showed:after proliferated for 4 to 5 days,myoblasts were indirectly stained with desmin antibody. The result showed myoblasts were of positive stain.The above results demonstrate that cultured cells were myoblasts.3.Effects of MOO on TGF-β2Results of TGF-β2 in immunocytochemistry showed:Compared with blank group,expression of TGF-β2 was increased in rest 5 group(P＜0.05) and rised with increasing Moo dose;Compared with 5-Aza-dc group,the rest group was up-regulated except low dose group(P＜0.05);Compared with combined group, 5-Aza-dc group's was decreased(P＜0.05).4.Effects of MOO on Smad4Results of Smad4 in immunocytochemistry showed:Compared with blank group, expression of Smad4 was increased in rest 5 group(P＜0.05).5.Effects of MOO on p-Smad2/3Results of p-Smad2/3 in Western Blotting showed:Compared with blank group, the expression of p-Smad2/3 was up-regulated slightly in MOO 100μg/ml and down-regulation rised with increasing Moo dose,of which,MOO 500μg/ml(P＜0.05); the expression of p-Smad2/3 was down-regulated in 5-Aza-dc group(P＜0.05) but up-regulated slightly in combined group.Compared with 5-Aza-dc group,expression of p-Smad2/3 was up-regulated in MOO 100μg/ml group and combined group(P＜0.05) but was down-regulated in Moo 500μg/ml.Compared with combined group, expression of p-Smad2/3 was decreased in 5-Aza-dc group and Moo 300μg/ml (P＜0.05).6.Effects of MOO on Smad7Results of Smad7 in Western Blotting showed:Compared with blank group, expression of the rest groups' Smad7 had difference obviously(P＜0.05).of which,the expression of Smad7 was inhibited in the MOO 100μg/ml,and rised with increasing Moo dose.the expression of Smad7 was down-regulated in the 5-Aza-dc group and combined group;Compared with 5-Aza-dc group,expression of Smad7 was up-regulated in MOO 500μg/ml group(P＜0.05) and was inhibited in Moo 100μg/ml(P＜0.05);Compared with combination group,the same results as compared with 5-Aza-dc group. Conclusions:1 MOO can up-regulate expression of TGF-β2 in dose-dependent.2 MOO can influence expression of downstream signal transcriptional factors Smads of TGF-β2.of which,Up-regulate expression of Smad4;Different dose show bi-directional influences to p-Smad2/3 and Smad7.3 MOO 300μg/ml combining 5-Aza-dc shows independence penomenon to expression of TGF-β2,Smad4 and Smad7.But synergism to expression of p-Smad2/3.4 Effects of MOO on TGF-β2/Smads is one of importance mechanisms that MOO differentiated myoblasts into myocardial-like cells...