An Experimental Study On The Prevention Of Steroid-induced Osteonecrosis Of Femoral Head By SiRNA Inhibiting Expression Of PPARγGene

Steroid-induced osteonecrosis of the femoral head (ONFH) is a common disease and the number of patients is increasing year by year. 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 mankind health. No means can prevent onset of the disease to our knowledge at present. The majority of patients had to receive total hip replacement. Treatment and prevention of ONFH becomes a worldwide problem in the field of orthopedics. Peroxisome proliferator-activated receptor-y (PPARγ) is one of the nuclear hormone receptor, an adipogenic transcription factor involved in the differentiation of fat cells in adipose tissue and induction of adipocyte differentiation. It is does not express in the former fat cells but in adipose differentiation and before most of the fat gene expression. If there is no PPARγ, the preadipocytes are difficult to differentiate into fat cells. Cytological studies and animal experiments show that steroid can increase expression of PPARγmRNA, in the marrow stromal cells (MSCs) in human and rabbit bodies, and then directly induce the differentiation of MSCs into a large number of adipocytes and inhibit their osteogenic differentiation, causing adipose cell proliferation and hypertrophia as well as lipid deposition in osteocytes, fatty degeneration and dead of cells in femoral heads. PPARγis an important target gene of the incidence of steroid-induced osteonecrosis. Therefore we can assume that inhibition of PPARy expression in MSCs could help preventing MSCs from differentiating into adipocytes, making it possible that the prevention of femoral head necrosis. We used effective RNA interference (RNAi) technique, selected the high effective segment of siRNA targeting PPARγmRNA and constructed recombinant adenovirus vector for expressing siRNA. The vectors were infected to rabbit MSCs in vitro and the cells of femoral head in vivo. The preventive effects of siRNA adenovirus vector on steroid-induced ONFH were observed by the methods of molecular biology, cytology and zoology, providing a reliable scientific basis for prevention of femoral head necrosis in the incidence of the initial link.Partâ… Preparation of the PPARγsiRNA adenovirus shuttle vector and recombinant adenovirus1. MethodsAccording to the principles of siRNA design and the PPARγgene sequence (GenBank Accession No. NM₀01082148), three duplexes of specific siRNA sequences and one nonspecific sequence were synthesized. And the corresponding hairpin RNA (shRNA) oligonucleotide primers was synthesized. Xhoâ… and Speâ… restriction enzyme digestion sites were added in both ends. Six synthesized oligonucleotides (PPARγ-971-1, PPARγ-971-2, PPARγ-1253-1, PPARγ-1253-2, PPARγ-1367-1 and PPARγ-1367-2) were dissolved completely in 50μL ultrapure water. The annealed PPARy and short hairpin DNA control oligos were loaded onto 2.0% agarose gel electrophoresis. Thereafter, products were purified by DNA gel extraction kit according to manufacture's instructions. Annealed products were ligated into shuttle vector 1.0-CMV vector. The recombinant vector was transformed into DH5a competent cells routinely. Positive clones of shuttle vector 1.0-CMV and DNA plasmids were extracted and were under restriction enzyme digestion and sequencing. Then we got the confirmed recombinant adenovirus shuttle vector:shuttle vector 1.0-CMV-971, shuttle vector 1.0-CMV-1253, shuttle vector 1.0-CMV-1367 and shuttle vector 1.0-CMV-Con.After DNA sequencing, the confirmed plasmids were extracted by plasmid DNA mini-preparation kit according to manufacture's instructions (AXYGEN, U.S.A.), and were identified by 1% agarose gel electrophoresis. The plasmid concentration was evaluated by ultraviolet spectrophotometer. Transfection with shuttle vector 1.0-CMV-971, shuttle vector 1.0-CMV-1253 and shuttle vector 1.0-CMV-1367 plasmids was carried out by using the LipofectamineTM 2000 transfection reagent. The DNA-LipofectamineTM 2000 mixture was used for transfectionn of HEK293 cells. Four or five days after transfection, adenovirus was collected when cytopathic effect (CPE) of virus was over 80%. Virus purification was carried out using adenovirus purification kit according to manufacture's instructions (Biomiga, U.S.A.). Finally we got adeno 1.0-CMV-971, adeno 1.0-CMV-1253, adeno 1.0-CMV-1367å’Œadeno 1.0-CMV-con, virus titers were determined by plaque assay.Results:2.1 Preparation of the PPARy siRNA adenovirus shuttle vector After the annealing of single-strand DNA oligo encoding short hairpin RNAs (shRNA) sequence, products were identified by gel electrophoresis. Then, these products were ligated into shuttle vector 1.0-CMV vector and the recombinant vectors of shuttle vector 1.0-CMV-971, shuttle vector 1.0-CMV-1253 and shuttle vector 1.0-CMV-1367 were constructed. These recombinant vectors were further identified by sequencing. DNA sequencing confirmed the insertion sequence and design of sequences were identical.2.2 Preparation of recombinant adenovirusFour or five days after transfection, adenovirus was collected when cytopathic effect (CPE) of virus was over 80%. After purification, the virus titer was higher than 1010PFU/mL. That meant high titer of adenovirus carrying PPARysiRNA was successfully packaged.Partâ…¡. Interfere effect of siRNA adenovirus vector to adipogenic differentiation in MSCs of rabbit1 MethodsMSCs were firstly obtained and nurtured from New Zealand Rabbit. The experiment was divided into 6 groups. Group S1, S2 and S3 (siRNAl, S1,siRNA2, S2,siRNA 3, S3):the cells were treated with steroid and packed with adeno 1.0-CMV-1,2 and 3 (pSilencerTM adeno 1.0-CMV-971, pSilencerTM adeno 1.0-CMV-1253, pSilencerTM adeno 1.0-CMV-1367). Group Con (Control 1):the cells were treated with steroid and adenovirus packed with adeno 1.0-CMV-Con (pSilencerTM adeno 1.0-CMV-con). Group M (Model, M):the cells were treated with steroid only. Group N (Normal, N), the cells were treated neither with alcohol nor with the vector of pAdeno-X adenovirus. The second generation of MSCs Will be seeded in 6-well plates, plus 1 x 105IU virus in each well. MSCs were infected with virus for 12 hours. Then complete high glucose dulbecco's modified eagle's medium (DMEM) medium was added into the well. At the same time the final concentration of 10-7mol/L of steroid was added into the medium of the groups above, which needed steroid inducing. The very concentration of steroid was added into the new medium every time when it was replaced. The state and growing condition of the MSCs were observed through the inverted microscope.On the 1rd,3th,5th and 7th day of the first steroid inducement, the expression of the PPARγmRNA,osteocalcin mRNA,Runx2 mRNA were determined using the method of TaqMan RT-PCR and the amount of PPARγ,osteocalcin Runx2 protein were determined using the method of Western-blot respectively. On the 14th day, the contents of triglyceride (TG) and alkaline phosphatase (ALP) activity in MSCs were determined by biochemical assay; the content of osteocalcin (OC) in the media were determined by radioimmunoassay. On the 21th day, the adipocytes induced by the steroid in the nurtured MSCs were stained with the Sudanâ…¢and counted under a microscope.2 Results2.1 Determination of mRNA expression of adipogenic and osteogenic differentiation gene of MSCs:2.1.1 Determination of mRNA expression of PPARγ,Osteocalcin and Runx2: On the 1st,3rd,5th and 7th day of steroid inducement, the expression level of PPARy mRNA of group M and Con were higher than that of group N (P<0.05), the expression level of PPARγmRNA of group S1, S2 and S3 were lower than that of group Con and group M (P<0.05). On the 7th day of steroid inducement, the expression level of osteocalcin mRNA and Runx2 mRNA of group M and Con were lower than that of group N (P<0.05), the expression level of osteocalcin mRNA and Runx2 mRNA of group S1, S2 and S3 were almost the same as group N. The difference had no statistical significance (P>0.05). The results showed that siRNA adenovirus vector could effectively inhibit the expression of PPARy mRNA within MSCs while maintaining the expression of osteocalcin mRNA and Runx2 mRNA in MSCs.2.2.2 Determination of protein expression of PPARγ,Osteocalcin andRunx2: On the 7th day of steroid inducement, the immunoblotting showed that the blot of PPARγprotein of group N was less than that of group M and Con, but it was clearer than that of group S1, S2 and S3. It indicated that siRNA was able to suppress the expression of PPARy protein in the MSCs. The blot of osteocalcin and Runx2 protein of group N was clearer, the same as group S1, S2 and S3, but the blot of osteocalcin and Runx2 protein of group M and Con wan less than group N. That was siRNA adenovirus vector could inhibit PPARγprotein expression levels and maintain the levels of osteocalcin and Runx2 protein expression within the MSCs.2.3 Determination of the amount of adipocytes and the content of TG in adipogenic differentiation of MSCs:On the 14th day of steroid inducement, the content of TG were determined, on the 21st day of steroid inducement, the amount of adipocytes were counted. Group M and Con had greater amount of adipocytes and TG than group N. The difference had statistical significance (P<0.05). Group S1, S2 and S3 had lesser amount of adipocytes and TG than group M and group Con (P>0.05), almost the same amount of adipocytes and TG as group N. The differences had no statistical significance (P>0.05).2.4 Determination of OC the media and the ALP activity in osteogenic differentiation differentiation of MSCs:On the 14th day of steroid inducement, group M and group Con had lower ALP activity and OC in the media than that of group N. The difference had statistical significance (P<0.05). Group S1, S2 and S3 had almost the same amount of OC in the media as and a bit lower ALP activity values than group N. The differences had no statistical significance (P>0.05).Partâ…¢The preventive effect of siRNA adenovirus vector on steroid-induced ONFH1 MethodsWe randomly divided 48 2～3month-old (weight,2.5±0.5 kg) New Zealand rabbits (experimental animal center, Henan Province) into 4 groups. Group N:each was administered 2ml normal saline into gluteal muscle, three days in a row. Group M:each was administered 20mg/kg dexamethasone into gluteal muscle, three days in a row. Group S:each was administered 20mg/kg dexamethasone into gluteal muscle, three days in a row, meanwhile the recombinant virus drops 25μ1 (3×109 IU) was injected into one of femoral head intravenously on the 1st day in the 1st,3rd and 6th week. Group Con:administrate steroid as the same amount and method as above. The independent siRNA virus was injected into the animal side of the femoral head. Each was administered 20mg/kg dexamethasone into gluteal muscle, three days in a row, meanwhile the recombinant virus drops was injected into one of femoral head intravenously on the 1st day in the 1st,3rd and 6th week. The animals in the experiment were sacrificed in batches on four and eight weeks periodically. The contents of cholesterol total (CHO) and TG in serum were determined by biochemical assay. The percentage of empty osteocyte lacunae, average diameter of the max adipocyte, trabeculace area fraction in femoral heads were determinated by the light microscope after the specimens of femoral head were stained with Hematoxylin and eosin (HE) and Sudanâ…¢. The expression of the PPARγmRNA,osteocalcin mRNA,Runx2 mRNA were determined using the method of TaqMan RT-PCR and the amount of PPARγ,osteocalcin,Runx2 protein were determined using the method of Western-blot respectively.2 Results2.1 Serology:4 and 8 weeks after the injection, the contents of CHO, TG in serum in group M, Con and S were higher than that of group N. The difference had statistical significance (P<0.05)2.2 The histomorphological changes of femoral heads:Histopathologically, the changes of group M and group Con were as follows:the necrotic foci mainly observed in the subchondral region of the femoral head, the hematopoietic tissue in the femoral head marrow decreased and the fatty tissue increased, fatty cells in the head increased and enlarged were observed. The trabeculace became thinner and sparse, and its area fraction reduced. The percentage of empty osteocyte lacunae increased and there were statistically significant differences compared with the results of group N (P<0.05). The percentage of empty osteocyte lacunae, average diameter of the the max adipocyte and trabeculace area fraction in femoral heads of group S, approximated to the results of group N (P>0.05).2.3 Determination of mRNA expression of adipogenic and osteogenic differentiation gene of MSCs:2.3.1 Determination of mRNA expression of PPARγ,Osteocalcin andRunx2: The expression level of PPARγmRNA of group M, Con were higher than that of group N. The difference had statistical significance (P<0.05). The expression level of PPARγmRNA of group S approximated to the results in group N. The differences had no statistically significance (P>0.05). The expression levels of osteocalcin mRNA and Runx2 mRNA of group M, Con were lower than that of group N. The difference had statistical significance (P<0.05). The expression levels of osteocalcin mRNA and Runx2 mRNA of group S approximated to the result in group N. The differences had no statistically significance (P>0.05).2.3.2 Determination of protein expression of PPARγ,Osteocalcin andRunx2: The immunoblotting showed that the blot of PPARγprotein of group N was less than that of group M and Con, but it was clearer than that of group S. It indicated that siRNA was able to suppress the expression of PPARy protein in the MSCs. The blot of osteocalcin and Runx2 protein of group N was clearer, the same as group S, but the blot of osteocalcin and Runx2 protein of group M and Con wan less than group N. That was siRNA adenovirus vector could inhibit PPARγprotein expression levels and maintain the levels of osteocalcin and Runx2 protein expression within the MSCs. Conclusions1. In this study, three target siRNA adenovirus vectors for rabbit PPARγmRNA were successfully constructed, and the adenoviruses were also packaged in vitro with high titer and inhibition effect to PPARγexpression of MSCs, especially in group 2.2. The experiment in vitro confirmed that siRNA adenovirus vectors were able to inhibit expression of PPARγmRNA, protein and steroid-induced adipogenesis in MSCs, thus maintained the character of differentiation of MSCs into osteoblastic.3. The experiment in vivo confirmed that siRNA adenovirus vectors was able to inhibit expression of PPARγmRNA and alcohol-induced adipogenesis in MSCs of femoral head, thus maintained its osteogenic differentiation. The preventive effect of siRNA adenovirus vector on alcohol-induced ONFH was observed by the methods of zoology and molecular biology.4. In this study, using siRNA technology by blocking PPARγgene expression in MSCs lays a foundation for the research on pathogenesis and genetic prevention and treatment of steroid-induced ONFH.