The Mechanism Of Co-activator On Osteogenic Differentiation In MG63 And The Effect Of A Kidney-tonifying Herbal Fufang On SRC-3 In OVX Rats

Postmenopausal osteoporosis is a systemic bone disease characterized by decreased bone mass, fragile bone tissue due to impaired bone matrix, increased risk of fragile bone fracture. The risk of postmenopausal osteoporosis is associated with advanced age and gender. It is common among postmenopausal women and elderly men. This study focuses on postmenopausal osteoporosis(PMO) due to lack of estrogen. Decreased ovarian function cause decreased estrogen and further lead to the decrease of estradiol, growth hormone, thyroid hormone, and increase of FSH and LH through hypothalamic-pituitary-gonadal axis. This resulted in the increase of bone absorption. On the cell level, bone loss is due to functional imbalance between osteoclast and osteoblast. Postmenopausal osteoporosis has high bone turnover with both active bone absorption and bone formation. But bone absorption is excessive while formation is inadequate. Studies show that both nuclear receptor ER and ERR-alpha involve with estrogen to regulate the bone metabolism. The banding of ligand and nuclear receptor cause nuclear receptor structure change and activation, this leads to up or down regulation of gene expression. Nuclear receptor’s transcriptional activation, which is also controlled by coactivator, is important for the gene expression of nuclear receptors. Coactivator SRC regulate ER’activation to influence bone metabolism. PGC-1 and ERRalpha interact to control bone health.Bone tissue is complex human system. It’s physiological activities involve different cells, hormones, nerve sysytem and cytokine. This article further discussed the effects and mechanism of co-activator SRC-3 and PGC-la in bone metabolism. And also the possible mechanism of prevention postmenopausal osteoporosis using kidney-tonifying herbs. This study provides theoretical support for Chinese medicine at cell level, provides another option for the prevention and treatment of postmenopausal osteoporosis.Objectives:To study the mechanisms of coactivator SRC-3, PGC-la effects on MG63 cell differentiation and observe the effect of kidney-tonifying fu-fang herbs on bone density, metabolism and SRC-3.Method:1 Cell studyThis study used adenovirus silencing or overexpression technology to study the effects of coactivator SRC-3 and PGC-la on the differentiation and growth of MG63. MTT was used to detect cell proliferation ability, ALP was used to detect osteogenic ability of osteoblast. Real-time PCR was used to detect the MG63 cell gene expression ERRa, Runx2, Osteocalcin, Osterix mRNA levels. Use Western blot to detect cell MAPK signal channel P38MAKP, JNK and ERK1/2 level. At the same time, we applied PAPK channel block using real-time PCR and Western-blotting to detect the influence of SRC-3, PGC-la on MG63 cell differentiation and protein expression of MARK signal channel.2 Animal StudyDevelop postmenopausal osteoporosis model using OVX rats. Use kidney-tonifying herb granules to intervene. ALEN was used as positive control. DEXA test, ELISA and Real-time PCR were used to study the effect of the herb granules on rats left femur bone density, blood PINP, CTX, SRC-3 level, and SRC3 mRNA expression.Results:1 Adenovirus carrying SRC-3 and PGC-la regulate MG63 osteogenic differentiation.1.1 Construction and identification of human SRC-3 and PGG-la Gene silencing and over expression.Construction and identification of silencing group adenovirus:real-time PCR and Western-blot were used to find siSRC-3-3 and siPCG-la-1 sequence. Design and synthesize coresponsive shRNA sequence. Virus titrations are 0.98×109ifu/ml,1.3×109 ifu/mL and 1.95×109/ml. After infecting MG63, we use qPCR and west blot to verify the silencing effect of target gene and protein. It showed ShSRC-3 and shPGC-la carried by adenovirus have better silencing effects.Construction and identification of over-expression adenovirus:search SRC-3 and PGC-1a nuclear sequence, use cDNA as model to expand. Add multiple clone site, construct and regroup shuttle plasmid, collected pAD-SRC-3 pAD-PGC-la and pAD-Con virus titre:0.18×109 ifu/mL、0.43×109 ifu/mL �'� 0.89×109 ifu/ml. After infect MG63 cell, use qPCR and Western blot detected the existence of SRC-3 and PGC-1 a. This indicated SRC-3 and PGC-la carried by adenovirus can express normally.1.2 Silence or over-expression Adenovirus infected cell multiplication and osteogenic ability detection.MG63 proliferative ability:Silence group Ad-shSRC-3+Ad-shPGC-la has strongest inhibition followed by Ad-shPGC-la group than Ad-shSRC-3 group. In Over expression groups, pAd-SRC-3+pAd-PGC-la group has the strongest effect on MG63 proliferation ability. And it is stronger than the silence groups in 72h (P<0.05).ALP activity detection of MG63 osteogenic ability:after trasfection 24h and 48h, there are no difference between silence group and over expression group(P>0.05). But after 72h, there are different ALP activity(P<0.05). And pAd-SRC-3+pAd-PGC-la gourp ALP is higher than other group compared with Ad-shSRC-3+Ad-shPGC-la and pAd-Con group (P=.024, P=0.023). Other groups did not show statistic significance.MG63 cell osteogenesis related genes ERRα、OPN、Runx2 mRNA Expression: After Transfection MG63 cell 48h, total DNA was abstracted, the level of ERRa、 Runx2、Osteocalcin and Osterix mRNA in pAd-SRC-3+pAd-PGC-la group are higher than any other group. (P<0.01, P< 0.001, P<0.001, P<0.001). Ad-shSRC-3+Ad-shPGC-la in silence group was the lowest. OPN mRNA expression level among each groups has no difference (P>0.05)The level P38MAPK、JNK、ERK1/2 in MG63 cell:after transinfect ion MG63 48h then abstract total cell protein. pAd-SRC-3+pAd-PGC-la group P-P38MAPK protein expression level in over-expression group is significant higher than pAD-Con group, pAD-SRC-3 group (p<0.01). There are no significant difference between p38MAPK、JNK、P-JNK、ERK1/2、P-ERK1/2 groups.1.3 MAPK signaling pathway inhibitor intervene the osteogenesis ability of silence or over-expression adenovirus transfection of MG63.ALP activity of MG63 cell after intervened by 3 MAPK signaling pathway inhibitor:ALP level in pAd-SRC-3+pAd-PGC-1a group is higher than all other groups (P<0.01). But compared with pAd-SRC-3+pAd-PGC-1a+SP600125 and pAd-SRC-3+pAd-PGC-la+PD98059, there are no difference (P>0.05). pAd-SRC-3+ pAd-PGC-1a+SB203580 and pAd-SRC-3+pAd-PGC-1a (P<0.001) This shows that the intervention of Adenovirus transfection MG63 can promote ALP activity. SB203580 can inhibit the ALP activity. But the intervention of SP600125 and PD98059 has no significant effect.Phosphorylation level of P38MAPK、JNK、ERK1/2 in MG63 cell after intervention by MAPK signaling pathway inhibitor:total cell protein 4 was extracted 8h after inhibitor intervene MG63 cell. The protein expression P-P38MAPK level in pAd-SRC-3+pAd-PGC-la group is significant higher than control group. (P<0.01) but pAd-SRC-3+pAd-PGC-la+SB203580 group and control group has no difference (P>0.05) Compared each group, p38MAPK、JNK、P-JNK、 ERK1/2、P-ERK1/2 protein (P>0.05) protein expression levels are not statistically different. (p>0.05). This shows that pAd-SRC-3+pAd-PGC-la group can promote P-p38MAPK protein expression, and p38MAPK signaling pathway inhibitor can supress the expression.The expression of ERRα、Runx2、Osteocalcin、Osterix mRNA in MG63 cell after intervention by r p38MAPK inhibitor SP600125:Extract total RNA, SRC-3、 PGC-1a 48h after MG63 cell processed by P38MAPK inhibitor SB203580. This show it can promote MG63 cell ERRa、Osteocalcin Osterix, mRNA expression level. And P38MAPKSB203580 signaling pathway inhibitor can block the expression. But SRC-3、PGC-1a co-overexpression have no effect on MG63 Runx2 mRNA expression.2. The Effect of Kidney tonifying herb Fufang on SRC-3 and PGC-1α expression of osteoporosis of overiectomized rats.2.1 Kidney-tonifying herb Fufang can increase bone density of overiectomized rats.Bone density changes after 12 weeks intervention:OVX group femur bone density is lower than Sham Group (*P<0.001). This indicated the success of osteoporosis model. BSZG group and ALEN group bone density is higher than OVX group (P<0.01). But BSZG group and ALEN group bone density have no significant difference (P>0.05). This indicates that BSZG group can effectivly prevent osteoporosis in OVX model rats. And it has the same effect as the ALEN group.2.2 The Effect of kidney-tonyfying herb Fufang on Serological indicators of overiectomized rats.OVX group blood PINP、CTX level is higher than Sham group (P<0.001). BSZG group and ALEN group blood PINP、CTX level both are lower than the OVX group (P<0.001). But the difference between BSZG group and ALEN group blood PINP、 CTX levels have no statistical significance (P>0.05)This indicates that both BSZG group and ALEN group can decrease PINP、 CTX level in overiectomized rats, and they have the same effect. OVX group blood SRC-3 level is lower than Sham group (P<0.001.) But BSZG group SRC-3 levels significantly higher than OVX group and ALEN group (P<0.001, P<0.05). This indicates that BSZG group can increase blood SRC-3 level in overiectomized rats, and it is better than ALEN group。2.3 The effects of kidney tonifying herb fu-fang on SRC-3 mRNA expression in overiectomized rats.SRC-3 mRNA level in the femur of overiectomized rats among OVX group, BSZG group and ALEN group are all lower than sham group (P<0.05). But BSZG group femur SRC-3 mRNA expression levels higher than OVX group (P<0.05). But between ALEN group and OVX group there is no difference (P>0.05). This indicates BSZG group can increase femur marrow SRC-3 mRNA expression level and it is better than ALEN group.Conclusion:It showed that SRC-3 and PGC-1α can be expressed in MG63 cells. After adenovirus mediate SRC-3 and PGC-1α double gene combine over-expression, and transfection of MG63, it promoted MG63 proliferation, activated interior ERR a gene expression and P38MAPK path, which lead to the increase of ALP activity and osteogenic specific gene expression of Runx2, Osteocalcin、Osterix. Thus it promoted the osteogenic differentiation of MG63. While double genes silence of ShSRC-3 and shPGC-la can inhibit osteogenic differentiation.It is possible that kidney tonifying herb Fu-fang can increse marrow SRC-3 mRNA expression level so that it increased bone density and decreased bone turnover level.