金松雙黃酮抗骨質(zhì)疏松癥作用的機(jī)制研究
發(fā)布時(shí)間：2018-10-24 21:31
【摘要】：骨是一種動(dòng)態(tài)變化的組織。破骨細(xì)胞的骨吸收活性和成骨細(xì)胞的骨形成活性,共同維持骨的動(dòng)態(tài)平衡。這種平衡被打破容易導(dǎo)致骨疾病的發(fā)生。破骨細(xì)胞的數(shù)量過(guò)多或是骨吸收活性過(guò)強(qiáng),將會(huì)導(dǎo)致骨質(zhì)流失,容易引起骨質(zhì)疏松癥。目前骨質(zhì)疏松的治療方案主要是通過(guò)降低骨吸收或促進(jìn)骨形成來(lái)實(shí)現(xiàn)的。有報(bào)道稱金松雙黃酮(Sc)具有保護(hù)成骨細(xì)胞,促進(jìn)骨形成的作用,但是對(duì)破骨細(xì)胞的影響和作用機(jī)制尚未有報(bào)道。本研究旨在探討金松雙黃酮對(duì)破骨細(xì)胞的影響及其作用機(jī)制,以及對(duì)LPS(脂多糖)誘導(dǎo)的小鼠骨丟失模型的影響。本研究通過(guò)體外分離培養(yǎng)小鼠骨髓單核細(xì)胞(BMMs),并用細(xì)胞因子M-CSF和RANKL誘導(dǎo)分化成破骨細(xì)胞。用不同濃度的金松雙黃酮溶液刺激破骨細(xì)胞,探討對(duì)破骨細(xì)胞的生存、分化和功能的影響。采用Real-time PCR和Westernblotting方法檢測(cè)金松雙黃酮對(duì)破骨細(xì)胞相關(guān)基因的轉(zhuǎn)錄以及對(duì)NF-κB和MAPK信號(hào)通路的影響。結(jié)果顯示金松雙黃酮能夠抑制破骨細(xì)胞的分化成熟和骨吸收活性,并有劑量依賴性。同時(shí)還能夠抑制破骨細(xì)胞相關(guān)基因,如Cts K、MMP-9、TRAP、NFATc1和C-Fos的轉(zhuǎn)錄,抑制NF-κB信號(hào)通路的活化,但是對(duì)MAPK信號(hào)通路的磷酸化并無(wú)作用。為了進(jìn)一步研究金松雙黃酮對(duì)骨質(zhì)疏松癥的治療效果,我們使用LPS腹腔注射建立了小鼠骨丟失模型。LPS是革蘭氏陰性菌外壁的重要組成部分,促進(jìn)炎性因子的產(chǎn)生,如TNF-α、IL-6、IL-1β等,造成體內(nèi)炎性環(huán)境,促進(jìn)破骨細(xì)胞的形成和活化,導(dǎo)致炎性骨丟失。在本研究中,小鼠被隨機(jī)分為對(duì)照組(SHAM)、模型組(LPS)和加藥組(LPS+Sc)。小鼠每隔兩天注射一次7.5μg/g體重量的LPS或鹽水,在LPS注射前1h,給予藥物或鹽水。LPS首次注射后第八天安樂(lè)死處死小鼠,收集小鼠腿骨。采用TRAP染色、HE染色和Micro-CT方法,檢測(cè)金松雙黃酮對(duì)破骨細(xì)胞的活性以及骨組織結(jié)構(gòu)的影響。結(jié)果顯示,金松雙黃酮在體內(nèi)能夠抑制破骨細(xì)胞的形成,明顯改善由LPS引起的骨小梁斷裂、骨密度降低。通過(guò)以上實(shí)驗(yàn)得出,在體外,金松雙黃酮通過(guò)抑制NF-κB信號(hào)通路活化,下調(diào)下游破骨細(xì)胞分化關(guān)鍵轉(zhuǎn)錄因子C-Fos和NFATc1的表達(dá)水平,進(jìn)而抑制破骨細(xì)胞分化標(biāo)志性基因的表達(dá),最終使得破骨細(xì)胞的分化成熟和骨吸收功能得到抑制。在體內(nèi),金松雙黃酮同樣可以抑制破骨細(xì)胞的活性,改善由LPS引起的骨丟失癥狀。體內(nèi)和體外實(shí)驗(yàn)結(jié)果表明,金松雙黃酮具有抑制破骨細(xì)胞分化及骨吸收功能的作用,可以作為治療骨質(zhì)疏松癥的候選藥物進(jìn)一步研究。
[Abstract]:Bone is a kind of dynamic tissue. The bone resorption activity of osteoclasts and the osteogenic activity of osteoblasts together maintain the dynamic balance of bone. This balance is broken and can easily lead to bone disease. Excessive number of osteoclasts or excessive bone resorption may lead to bone loss and osteoporosis. The current treatment of osteoporosis is mainly achieved by reducing bone resorption or promoting bone formation. It has been reported that Jin Song bisflavone (Sc) can protect osteoblasts and promote bone formation, but the effect of Jin Song on osteoclasts and its mechanism have not been reported. The purpose of this study was to investigate the effect of Jin Song bisflavone on osteoclasts and its mechanism, and on the bone loss model induced by LPS (lipopolysaccharide) in mice. In this study, mouse bone marrow monocytes (BMMs),) were isolated and cultured in vitro and induced to differentiate into osteoclasts by cytokines M-CSF and RANKL. The effects of different concentrations of Jin Song bisflavone solution on the survival, differentiation and function of osteoclasts were studied. Real-time PCR and Westernblotting were used to detect the effect of Jin Song bisflavone on the transcription of osteoclast related genes and on NF- 魏 B and MAPK signaling pathway. The results showed that Jin Song could inhibit osteoclast differentiation, maturation and bone resorption in a dose-dependent manner. It also inhibited the transcription of osteoclast related genes, such as Cts, MMP-9, TRAPP, NFATc1 and C-Fos, and inhibited the activation of NF- 魏 B signaling pathway, but had no effect on the phosphorylation of MAPK signaling pathway. In order to further study the therapeutic effect of Jin Song bisflavone on osteoporosis, we established a model of bone loss in mice by intraperitoneal injection of LPS. LPS is an important component of the outer wall of Gram-negative bacteria and promotes the production of inflammatory factors, such as TNF- 偽, IL-6,IL-1 尾, etc. Cause inflammatory environment, promote osteoclast formation and activation, leading to inflammatory bone loss. In this study, mice were randomly divided into control group (SHAM), model group (LPS) and admixture group (LPS Sc). Mice were injected with 7.5 渭 g / g LPS or saline every two days, and then given drugs or saline one hour before LPS injection. The mice were euthanized on the eighth day after the first LPS injection, and the leg bones of the mice were collected. The effects of Jin Song bisflavone on osteoclast activity and bone structure were detected by TRAP staining, HE staining and Micro-CT staining. The results showed that Jin Song could inhibit the formation of osteoclasts in vivo, improve the trabecular fracture induced by LPS, and decrease bone mineral density (BMD). From the above experiments, it can be concluded that in vitro, Jin Song bisflavone can inhibit the activation of NF- 魏 B signaling pathway and down-regulate the expression of C-Fos and NFATc1, the key transcription factors of downstream osteoclast differentiation, and then inhibit the expression of the signature genes of osteoclast differentiation. Finally, osteoclast differentiation, maturation and bone resorption were inhibited. In vivo, Jin Song bisflavone also inhibited the activity of osteoclasts and improved the symptoms of bone loss caused by LPS. The results of in vivo and in vitro experiments showed that Jin Song bisflavone could inhibit osteoclast differentiation and bone resorption and could be used as a candidate drug for the treatment of osteoporosis.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R580
[Abstract]:Bone is a kind of dynamic tissue. The bone resorption activity of osteoclasts and the osteogenic activity of osteoblasts together maintain the dynamic balance of bone. This balance is broken and can easily lead to bone disease. Excessive number of osteoclasts or excessive bone resorption may lead to bone loss and osteoporosis. The current treatment of osteoporosis is mainly achieved by reducing bone resorption or promoting bone formation. It has been reported that Jin Song bisflavone (Sc) can protect osteoblasts and promote bone formation, but the effect of Jin Song on osteoclasts and its mechanism have not been reported. The purpose of this study was to investigate the effect of Jin Song bisflavone on osteoclasts and its mechanism, and on the bone loss model induced by LPS (lipopolysaccharide) in mice. In this study, mouse bone marrow monocytes (BMMs),) were isolated and cultured in vitro and induced to differentiate into osteoclasts by cytokines M-CSF and RANKL. The effects of different concentrations of Jin Song bisflavone solution on the survival, differentiation and function of osteoclasts were studied. Real-time PCR and Westernblotting were used to detect the effect of Jin Song bisflavone on the transcription of osteoclast related genes and on NF- 魏 B and MAPK signaling pathway. The results showed that Jin Song could inhibit osteoclast differentiation, maturation and bone resorption in a dose-dependent manner. It also inhibited the transcription of osteoclast related genes, such as Cts, MMP-9, TRAPP, NFATc1 and C-Fos, and inhibited the activation of NF- 魏 B signaling pathway, but had no effect on the phosphorylation of MAPK signaling pathway. In order to further study the therapeutic effect of Jin Song bisflavone on osteoporosis, we established a model of bone loss in mice by intraperitoneal injection of LPS. LPS is an important component of the outer wall of Gram-negative bacteria and promotes the production of inflammatory factors, such as TNF- 偽, IL-6,IL-1 尾, etc. Cause inflammatory environment, promote osteoclast formation and activation, leading to inflammatory bone loss. In this study, mice were randomly divided into control group (SHAM), model group (LPS) and admixture group (LPS Sc). Mice were injected with 7.5 渭 g / g LPS or saline every two days, and then given drugs or saline one hour before LPS injection. The mice were euthanized on the eighth day after the first LPS injection, and the leg bones of the mice were collected. The effects of Jin Song bisflavone on osteoclast activity and bone structure were detected by TRAP staining, HE staining and Micro-CT staining. The results showed that Jin Song could inhibit the formation of osteoclasts in vivo, improve the trabecular fracture induced by LPS, and decrease bone mineral density (BMD). From the above experiments, it can be concluded that in vitro, Jin Song bisflavone can inhibit the activation of NF- 魏 B signaling pathway and down-regulate the expression of C-Fos and NFATc1, the key transcription factors of downstream osteoclast differentiation, and then inhibit the expression of the signature genes of osteoclast differentiation. Finally, osteoclast differentiation, maturation and bone resorption were inhibited. In vivo, Jin Song bisflavone also inhibited the activity of osteoclasts and improved the symptoms of bone loss caused by LPS. The results of in vivo and in vitro experiments showed that Jin Song bisflavone could inhibit osteoclast differentiation and bone resorption and could be used as a candidate drug for the treatment of osteoporosis.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R580
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