The Effect On The Differentiation Of Alcohol-induced Osteonecrosis Of Femoral Head Of Rats By MiR-548d-5p Targetedly Regulating The Expression Of PPARγ Gene

Background and PurposeAlong with the improvement of economic level, more and more people drink. And alcohol-induced osteonecrosis of the femoral head(AONFH), with the increasing consumption of alcohol, becomes one of the orthopeadics diseases and the number of patients tends to increase year by year. What’s more, About 80% of them without effective treatment will suffer femoral head collapse and damage to joint function. It can cause physical disability in high rate and seriously threaten people’s health and life. At this time, there is no clinical significance for Conservative treatment. Therefore, it is strikingly important to prevent AONFH.Nowadays, there are varieties of theories about the pathogenesis of femoral head necrosis, such as intravascular coagulation theory, fat embolism theory, high pressure in bone and venous stasis theory, low coagulation high fibrinolysis theory, theory of osteoporosis,etc. Further studies find it that genes in the experimental animal heads down-regulate osteogenesis differentiation and up-regulate cells adipogenesis differentiation, which is induced by alcohol. BMCSs, instead of osteogenic differentiation, are also induced into adipogenic differentiation. Then it causes lipid deposition, pressure increase and blood decrease in the femoral heads, which causes ischemic necrosis of femoral head. PPARγ is an adipogenic transcription factor inducing adipocyte differentiation. It does not express in the former fat cells but in adipose differentiation and expresses prior to most of the fat gene. Moreover, preadipocytes are difficult to differentiate into fat cells without PPARγ.So we infer if BMSCs are prevented from differentiating into adipocytes by inhibition of PPARγ expression, it is possible that we could prevent osteonecrosis of the femoral head. This study intends to transfer mi R-548d-5p segment into rats’ BMSCs in vitro with electroporation method, which prevents the targeted genes PPARγ expression and inhibits BMSCs from differentiating into adipocytes. As a result, it can, in early period, prevent the process that alcohol induces BMSCs to differentiate into fat cells, and the occurrence of AONFH in the initial key links of its pathogenesis. Material and MethodsBMSCs were obtained and nurtured from male rats. The experiment was divided into 5 groups: Group N(Normal, N), the cells were not treated with alcohol and/or genes. Group M(Model, M): the cells were treated with alcohol only. Group NC(non-consequence, NC): the cells were treated with alcohol and non-consequence. Group G(Gene, G): the cells were treated with alcohol and mi R-548d-5p.After treated according to the grouping conditions, the second generation of BMSCs were seeded in 6-well plates, filled alcohol and the complete low glucose dulbecco’s modified eagle’s medium(DMEM) in each well and kept the final alcoholic concentration 0.09mol/L whenever DMEM was replaced. The state and form of the BMSCs were observed carefully through the inverted microscope.On the 3rd and 7th day of the alcohol inducement, the expression of PPARγ m RNA、BMP-2 m RNA、Runx2 m RNA and their corresponding proteins were respectively determined using the method of Taq Man RT-PCR and Immunocytochemistry. On the 14 th day of the alcohol inducement, the activity of alkaline phosphatase(ALP) and Triglyceride(TG) contents in BMSCs were respectively determined by Enzyme-linked immunosorbent assay(ELISA). At the same time, the adipocytes induced by alcohol in BMSCs nurtured were stained with oil red O and counted by microscopes. Results 1 Determination of m RNA expression and proteins of PPARγ、BMP-2 and Runx2 in BMSCsOn the 3rd and 7th day of the alcohol inducement, the expression level of PPARγ m RNA of group M and group NC were higher than that of group N(P<0.05), the expression level of PPARγ m RNA of group G were lower than that of group M and group NC(P<0.05). On the 3rd and 7th day of the alcohol inducement, compared with the expression level of group N, that of BMP-2 m RNA and Runx2 m RNA of group M and NC were lower(P<0.05), the expression level of BMP-2 m RNA and Runx2 m RNA of group G were almost the same as group N, and their difference had no statistical significance(P>0.05). The results of Immunocytochemistry method indicated that the expression level of corresponding proteins in group N and group G were higher than those of group M and group NC. The difference had statistical significance(P<0.05). 2 Determination the activity of ALP and the contents of TG in the differentiation of BMSCsOn the 14 th day of the alcohol inducement, group M and group NC had lower ALP activity and higher TG than those of group N. Their difference had statistical significance(P<0.05). Group G had almost the same activity values ALP and amount of TG than those of group N. The differences had no statistical significance(P>0.05). 3 The oil red O staining of BMSCs and determination of the amount of adipocytesOn the 14 th day of the alcohol inducement, the amount of adipocytes were counted after stained by oil red O. Group M and NC had greater amount of adipocytes than group N. Their difference had statistical significance(P<0.05). Group G had lesser amount of adipocytes than group M and group NC(P<0.05), and almost the same amount of adipocytes as group N. Their differences had no statistical significance(P>0.05). Conclusions 1 The experiment in vitro proved that mi R-548d-5p were able to inhibit expression of PPARγ m RNA and alcohol-induced adipogenesis in BMSCs, thus maintained the character of differentiation of BMSCs into osteoblasts. The preventive effect of mi R-548d-5p on AONFH was observed by the methods of molecular and biochemistry biology. 2 In this study, using mi R-548d-5p by blocking PPARγ gene expression in BMSCs may lay a new foundation for the research on genetic prevention or treatment of AONFH.