金松雙黃酮抗骨質疏松癥作用的機制研究
發(fā)布時間：2018-10-24 21:31
【摘要】：骨是一種動態(tài)變化的組織。破骨細胞的骨吸收活性和成骨細胞的骨形成活性,共同維持骨的動態(tài)平衡。這種平衡被打破容易導致骨疾病的發(fā)生。破骨細胞的數量過多或是骨吸收活性過強,將會導致骨質流失,容易引起骨質疏松癥。目前骨質疏松的治療方案主要是通過降低骨吸收或促進骨形成來實現的。有報道稱金松雙黃酮(Sc)具有保護成骨細胞,促進骨形成的作用,但是對破骨細胞的影響和作用機制尚未有報道。本研究旨在探討金松雙黃酮對破骨細胞的影響及其作用機制,以及對LPS(脂多糖)誘導的小鼠骨丟失模型的影響。本研究通過體外分離培養(yǎng)小鼠骨髓單核細胞(BMMs),并用細胞因子M-CSF和RANKL誘導分化成破骨細胞。用不同濃度的金松雙黃酮溶液刺激破骨細胞,探討對破骨細胞的生存、分化和功能的影響。采用Real-time PCR和Westernblotting方法檢測金松雙黃酮對破骨細胞相關基因的轉錄以及對NF-κB和MAPK信號通路的影響。結果顯示金松雙黃酮能夠抑制破骨細胞的分化成熟和骨吸收活性,并有劑量依賴性。同時還能夠抑制破骨細胞相關基因,如Cts K、MMP-9、TRAP、NFATc1和C-Fos的轉錄,抑制NF-κB信號通路的活化,但是對MAPK信號通路的磷酸化并無作用。為了進一步研究金松雙黃酮對骨質疏松癥的治療效果,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,促進炎性因子的產生,如TNF-α、IL-6、IL-1β等,造成體內炎性環(huán)境,促進破骨細胞的形成和活化,導致炎性骨丟失。在本研究中,小鼠被隨機分為對照組(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,檢測金松雙黃酮對破骨細胞的活性以及骨組織結構的影響。結果顯示,金松雙黃酮在體內能夠抑制破骨細胞的形成,明顯改善由LPS引起的骨小梁斷裂、骨密度降低。通過以上實驗得出,在體外,金松雙黃酮通過抑制NF-κB信號通路活化,下調下游破骨細胞分化關鍵轉錄因子C-Fos和NFATc1的表達水平,進而抑制破骨細胞分化標志性基因的表達,最終使得破骨細胞的分化成熟和骨吸收功能得到抑制。在體內,金松雙黃酮同樣可以抑制破骨細胞的活性,改善由LPS引起的骨丟失癥狀。體內和體外實驗結果表明,金松雙黃酮具有抑制破骨細胞分化及骨吸收功能的作用,可以作為治療骨質疏松癥的候選藥物進一步研究。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R580
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