Vitamin A1 Promotes The Differentiation Of Mouse C2C12 Myoblasts Through The Formation Of Retinoic Cid By DHRS3

Skeletal muscle development is an important process,which is regulated by a variety of genes related to muscle differentiation.Studies have shown that exogenous nutrients can also promote muscle cell differentiation,such as vitamins,fatty acids and amino acids.Vitamin A has two kinds of vitamin A1 and vitamin A2,and their physiological functions are similar,in which vitamin A1 mainly exists in the retina of animal liver,blood and eyeball,also known as retinol(VA1).VA1 is a fat-soluble alcohol nutrient,which plays an important role in maintaining the normal growth and development of bones.It has been reported that VA1 may be involved in bovine muscle cell differentiation.However,it is not clear whether VA1 can regulate mouse muscle cell differentiation and its molecular mechanism.Here,we mainly explore the effect of VA1 on the differentiation of mouse C2C12 myoblasts and the molecular mechanism of its effect on skeletal muscle development.VA1 produces retinoic acid(RA): through two consecutive oxidation reactions.First,VA1 is reversibly oxidized to retinaldehyde(RAL);under the action of dehydrogenase / reductase(SDR)family member 3(DHRS3)and then RAL is irreversibly oxidized to RA.As a highly conserved member of the short-chain alcohol dehydrogenase / reductase superfamily,DHRS3 is a reversible enzyme and plays an important role in the metabolism of VA1.However,there are few reports on the expression and function of DHRS3 in mouse muscle cells.The main purpose of this study was to explore the effects of exogenous VA1,RAL and RA on the differentiation of C2C12 myoblasts;to explore the expression of DHRS3 in the process of C2C12 myoblast differentiation and in vivo muscle cell differentiation;to explore the effect of DHRS3 expression on mouse C2C12 myoblast differentiation;to explore the relationship between the mechanism of VA1 promoting mouse C2C12 myoblast differentiation and DHRS3.The main results are as follows:1.By adding VA1,RAL and RA,to the differentiation medium of mouse C2C12 myoblasts and using Western blotting and immunofluorescence techniques,the effects of exogenous VA1,RAL and RA on the differentiation of mouse C2C12 myoblasts were studied.The results showed that compared with the respective controls,the expression of muscle differentiation marker molecule Myo G was significantly increased,and the myotube fusion rate was significantly increased.Exogenous addition of VA1,RAL and RA could affect the differentiation of mouse C2C12 myoblasts in different days of differentiation.2.Mouse C2C12 myoblasts were induced to differentiate.Western blotting and immunofluorescence techniques were used to study the expression of DHRS3 during the differentiation of mouse C2C12 myoblasts.Western blotting results showed that the expression of DHRS3 increased with the increase of differentiation days of C2C12.The immunofluorescence results showed that DHRS3 was highly expressed in the differentiated myotubes,which was consistent with the result of Western blotting.3.In order to further explore the VA1 e of DHRS3 in muscle differentiation in vivo,we constructed a mouse muscle injury repair model and used Western blotting and immunohistochemical techniques to study the expression of DHRS3 in muscle differentiation in vivo.The results showed that in the process of muscle injury repair,the expression of DHRS3 gene increased gradually in the first three days of injury,reached the highest on the third day,and decreased gradually after the fifth day.In short,the expression of DHRS3 gene increased with the increase of muscle differentiation.4.By constructing activation inhibition vector,overexpression vector and interference vector,Western blotting and immunofluorescence techniques were used to study the effect of DHRS3 expression on the differentiation of mouse C2C12 myoblasts.The results showed that activation or overexpression of DHRS3 significantly increased the expression of Myo G,a marker of muscle differentiation,and significantly increased the rate of myotube fusion,while inhibition or interference with the expression of DHRS3 significantly decreased the expression of Myo G and significantly decreased the rate of myotube fusion.5.By adding VA1,RAL and RA,to the differentiation medium of mouse C2C12 myoblasts and interfering with the expression of DHRS3 with si RNA,the relationship between the effect on the differentiation of mouse C2C12 myoblasts and the expression of DHRS3 was studied by Western blotting and immunofluorescence techniques.The results showed that when the expression of DHRS3 was interfered,the expression of Myo G was significantly decreased in VA1 treated cells,and the myotube fusion rate of cells was significantly decreased.There was no significant change in RAL and RA groups.We studied the mechanism of the effect of VA1 on the differentiation of mouse C2C12 myoblasts by further adding VA1,and determining the content of RA by high performance liquid chromatography(HPLC).The results showed that the addition of VA1 and interference with the expression of DHRS3 could inhibit the differentiation induced by VA1 and reduce the content of RA.The above results show that exogenous addition of VA1,RAL and RA can promote the differentiation of C2C12 myoblasts in different degrees.The increase or decrease of DHRS3 expression promotes or inhibits the differentiation of C2C12 myoblasts respectively,suggesting that DHRS3 can affect the differentiation of C2C12 myoblasts.The addition of VA1 and interference with the expression of DHRS3 can inhibit the differentiation induced by VA1 and reduce the content of RA.These data suggest that VA1 may promote the differentiation of C2C12 myoblasts through the formation of RA catalyzed by DHRS3.Our results provide a theoretical basis for further analysis of the molecular mechanism of muscle development.