高原鼢鼠药用价值及分子机制研究
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摘要
高原鼢鼠(Myospalaxbaileyi)属仓鼠科(Cricetidae.)鼢鼠亚科,鼢鼠属(Myospalax),是栖息于青藏高原,危害草场和农田的地下啮齿动物。它是青藏高原草地生态系统中危害最为严重的啮齿类之一。由于其取食和挖掘活动,每年青藏高原的鼢鼠危害面积达数百万公顷,经济损失惨重。
塞隆骨为高原鼢鼠的干燥全骨架,是卫生部于1990年批准的(批准文号:〈90〉卫药试字Z33号)中药第一类新药材,研究发现其药理作用与虎骨有类似之处,主治风寒湿痹引起的肢体关节疼痛、肿胀、肌肤麻木、腰膝酸软(张宝琛,1996)。因此,是市场上许多成品中药的重要成分,如“塞隆风湿酒”、“塞雪风湿胶囊”、“晶珠风湿胶囊”等等。但其药用价值和作用机理的研究还不够深入,未研究具体的有效成分,对其作用机制、量效关系、时效关系和药代动力学探讨较少,其毒副作用未见报道。
本论文以高原鼢鼠为研究对象,提取塞隆骨的脂提物、醇提物、水提物和骨胶,制备塞隆骨水煎剂,探讨塞隆骨的药用价值;并利用生物酶水解高原鼢鼠肌肉,以期对高原鼢鼠进行综合开发利用,提高其经济价值和社会意义。
以大鼠成骨样细胞ROS17/2.8为细胞模型,通过MTT法、ALP活性检测、ALP染色、通过流式细胞仪测定成骨样细胞钙离子摄取,及用茜素红S法来对矿化结节计数观察对塞隆骨提取物成骨样细胞矿化功能的影响,在细胞水平上分析塞隆骨不同的提取组分对成骨样细胞ROS17/2.8增殖、分化、矿化的效果,并以激素类药物地塞米松作阳性对照。结果表明:塞隆骨脂提取物1×10~(-2)(g/ml)组、塞隆骨水煎液1×10~(-2)(g/ml)组、塞隆骨骨胶1×10~(-2)(g/ml)、1×10~(-2)(g/ml)组对成骨样细胞的增殖均较阳性、空白对照组强,且显著强于空白对照组
Myospalaxbaileyi, which belongs to Cricetidae family, Myospala subfamily and genera, is an underground rodent imperiling pasture and farmland on Qinghai-Tibet Plateau. It is one of the most devastating rodents for the Qinghai-Tibet Plateau grassland ecosystem. Because of its feeding and digging activity, every year it destroys millions of hektares of area and causes heavy economic loss to the Qinghai-Tibet Plateau.Sailonggu is the whole dehydrated skeleton of Myospalaxbaileyi and it' s the first-grade new medical material of Chinese traditional medicine authorized by the Ministry of Health in 1990. Researches have revealed that its pharmacal effect has similarities with tiger bones. Sailonggu can help relieve rheumatic pains, dispel pathogenic cold, remove dampness, induce menstruation, alleviate pain, and help build the liver and kidney. It' s also effective in the treatment of arthralgia, turgor, skin numbness, lassitude of waist and knee due to chill and damp arthralgia. Consequently it is an important component of many finished product of Chinese traditional medicine such as "Sailong wine for rheumatism" , "Saixue capsule for rheumatism" , "Jingzhu capsule for rheumatism" ,etc. Yet the research into its medical merit and action mechanism is not deep enough to study the concrete effective components.
Investigation into its action mechanism, quantity-effectiveness and time-effectiveness correlation and medical kinetics is deficient. Moreover, there is no report existing about its toxicity and sideeffect. This article uses Myospalaxbaileyi as the research target. Extract of Sailonggu from lipid, ethanol and water, and bone glue are made into watery decoction to probe into its medical merit; and by hydrolyzing Myospalaxbaileyi muscles with biological enzymes, we could expect overall development and utilization of Myospalaxbaileyi, improving it' s economic value and social significance.The experiment uses rat osteoblast ROS17/2.8 as the cell model, measures its calcium ion intake via MTT method, ALP activity detection and flow cytometry, observes the influence on its mineralization function by measuring the number of mineralization knots with alizarin red S method, analyses effectiveness of different Sailonggu extracts for the proliferation, differentiation and mineralization of osteoblast ROS17/2.8 on the cell level. The experiment also uses hormones medicine dexamethasone as a positive comparison. The outcome of the experiment proves that Sailonggu lipid extract 1×10-2 (g/ml) group, Sailonggu watery decoction 1×10-2 (g/ml) group, Sailonggu bone glue 1×10-1 (g/ml) and 1 ×10-2 (g/ml) group have more significant promotion to osteoblast' s proliferation when compared with blank comparison group and positive comparison group(dexamethasone group), with difference presenting significance value(p<0.05) if compared with the blank comparison group. When comparing the ALP staining result with the blank comparison group, it is shown that osteoblast cultured in those Sailonggu extracts are heavier stained by alkaline phosphatase. Positive cytoplasm stained by alkaline phosphatase should present light grey, sallow and grey black particles, the darker colour the higher content of the enzyme. Quantitative measurement of ALP indicates that after cultivation for 3 days, Sailonggu bone glue 1×10-1 (g/ml) and lipid extract 1 × 10-2 (g/ml)
groups have an obvious proliferation promotion (P<0.05). Those Sailonggu extract groups have stronger effect than the dexamethasone group and the treated cells have an apparently higher ALP level than the comparison group. The result indicates that Sailonggu extracts have an proliferation promotion to the R0S17/2. 8 rat osteoblast cultured in vitro; copared with blank comparison group, groups treated by medicine and positive comparison group obviously have more calcium knots (P <0.05) ; the medicine-treated groups and dexamethasone positive comparison group all have quite high calcium ingestion and 1×10-2(g/ml) bone glue group and lipid extract group showed quite high value which is significant compared with blank comparison group (P<0.05) . Sailonggu bone glue and lipid extract have a direct promotion for the mineralization of rat osteoblast cultured in vitro, and can improve osteoblast' s ability to form bones.The quantity and composition analysis of Myospalaxbaileyi flesh proved that its flesh has abundant protein, amino acid, unsaturated fatty acid, vitamine A, E, C,K and many kinds of microelement including Ca, Fe, K, Mn, Na and so on. The unsaturated fatty acid content is as high as 64. 14%, and linoleic acid(18:2) content is 28. 14%, higher than in any other animal fat. The EAA/TAA ratio reaches 39.65 % , close to FA0/WH0(1973) grading mode. The microelement content is characteristic in its high K and low Na content, according with the tendency of modern nourishment and health care.The optimum condition to decompose the flesh of Myospalaxbaileyi with papain is at the temperature of 55℃ for 6 hours, with the enzyme quantity at 2. 8%, solid-liquid ratio at 1:3.5 and pH at 6.0. Under this condition the hydrolysis extent of the flesh exceeds 48% and we could obtain 17 categories of amino acid including 8 essential ones that account for 46.58% of all the amino acid and have a rational proportion according with the human essential amino acid mode suggested by FAO/WHO, with an E/N value of 0. 92, larger than 0. 6 suggested by FAO/WHO. Because of the
high content of flavour amino acid, which occupies 26. 11% of the total amino acid, the digest is very delicious. In oder to probe into the physiological activity funtion of the digest, we employ the MTT method and neutral red colorimetric method to measure the effect it has on the proliferation of normal rat splenocyte and lymphocyte, phagocytic function of celiac macrophage and reticuloendothelial system, killing and toxicity property of NK cells, and the number of rat antibody-making splenocyte (IgM-PFC). Immunocompetence research on the zymolyte reveals that high and middle dosage (0. 4g/kg; 0. 2g/kg) have significant difference from the comparison group, especially the activity of the high dosage group. The zymolyte has a significant promotion on the cell and humoral immunity of normal rat as well as the killing activity and toxicity of NK cells.So as to further investigate the mechanism of Sailonggu' s promotion on osteoblast' s proliferation, differentiation and maturation, an osteoblast apoptosis experiment is designed, applying agarose gel electrophoresis and flow cytometer to study apoptosis of osteoblast. The result shows that osteoblast in Sailonggu extracts shows much fewer hypodiploid peeks. Agarose gel electrophoresis experiment proves that cells in blank comparison group present clear DNA Ladders whereas the Sailonggu ectract groups show no ladder-shaped DNA band. That means Sailonggu extracts can depress the apoptosis ratio of osteoblast and can postone its occurrence. The total RNA of osteoblast from the medicine-treated group and blank group are extracted to semi-quantif icationally determine the expression of IL-1 mRNA, IL-2 mRNA, IL-4 mRNA, IL-6 mRNA, IL-10 mRNA and IFN-γ mRNA with highly sensitive RT-PCR method. The extracts can promote the expression of a great variety of cell factor mRNA. As a whole, lipid extract and bone glue extract have stronger effect than dexamethasone and watery extract. These extracts could be effective in the bone metabolism through influencing the
expression of many cell factor genes.To find in the bone tissue the differently expressed genes related to curative effect of Sailonggu on osteoporosis after menopause, further illustrate the mechanism Chinese traditional medicine can prevent and cure osteoporosis, and to screen effective components in Sailonggu, we observe the influence of Sailonggu on the expression of OPG, OCN and cbfa genes in rat osteoblast cultured in vitro, exract the total mRNA in cells cultured for 3 days, and analyse the mRNA expression of OPG, OCN and cbfa using RT-PCR technique to make a semi-quantitative comparison. On the third day, the 3 genes all have some expression; relative to the blank comparison group, the Sailonggu extracts group and dexamethasone group increase the expression of OCN mRNA with extreme significance (P<0. 01), with Sailonggu extracts group having weaker effect than the dexamethasone group. Sailonggu extract groups, including lipid extract group 1×10-2, watery extract group 1×10-2 and bone glue group 1×10-3 , as well as dexamethasone group, all increase the expression of OPG mRNA(P<0. 01) while the dexamethasone group having stronger effect. Sailonggu watery extract group 1×10-2 and bone glue group 1×10-1 and dexamethasone group all increase the expression of OPG mRNA (P<0.05) with the lipid extract group 1×10-2 having stronger promotion than the dexamethasone group significantly (P<0. 01) .In order to study the anti-inflammation mechanism of different Sailonggu extracts, we do experiments with medicine filtration models which act on inflammation-related cell factors simultaneously. We adopt genetic transfection technique to obtain HepG2 cell line that can stably express recombinant protein, apply LPS to stimulate the formation of inflammation factors, qualitatively and quantitatively detect the fluorescence strength variation after treating the cell line with different extracts. And in these ways we probe into the feature of NF-kb activation caused by different Sailonggu extracts. Gelatingases in the
culture medium supernatant secreted by tumour cells and their active forms could be separated according to their different molecular weight by SDS-PAGE. SDS could be removed from protein by TritonX-100 so gelatingase can resume it' s protein degrading activity, which is used to evaluate the medicine' s ability to resist affection. We apply chemiluminescent COX inhibitor screening assay to detect influence on COX activity by different extracts to evaluate the medicine' s ability to resist affection and it turns out thai different Sailonggu extracts are capable of decreasing many inflammation factors with the lipid extraction having the best effect.To sum up, Sailonggu could promote proliferation and differentiation of rat osteoblast, the expression of OPG, OCN and cbfa mRNA, adjust the balance between osteoblast and osteoclast; and in these ways it can reconstruct the bones to prevent and cure osteoporosis. So it is suggested that Sailonggu could be a substitute for hormones to treat osteoporosis and has a great prospect to be developed into a new natural medical product to resist osteoporosis or bone absorption. Those Sailonggu extracts could be used as an antiinflammation Chinese traditional medicine, with the possibility to replace the tiger bone. And the zymolyte of Myospalaxbaileyi flesh has immunologic competence.
塞隆骨为高原鼢鼠的干燥全骨架,是卫生部于1990年批准的(批准文号:〈90〉卫药试字Z33号)中药第一类新药材,研究发现其药理作用与虎骨有类似之处,主治风寒湿痹引起的肢体关节疼痛、肿胀、肌肤麻木、腰膝酸软(张宝琛,1996)。因此,是市场上许多成品中药的重要成分,如“塞隆风湿酒”、“塞雪风湿胶囊”、“晶珠风湿胶囊”等等。但其药用价值和作用机理的研究还不够深入,未研究具体的有效成分,对其作用机制、量效关系、时效关系和药代动力学探讨较少,其毒副作用未见报道。
本论文以高原鼢鼠为研究对象,提取塞隆骨的脂提物、醇提物、水提物和骨胶,制备塞隆骨水煎剂,探讨塞隆骨的药用价值;并利用生物酶水解高原鼢鼠肌肉,以期对高原鼢鼠进行综合开发利用,提高其经济价值和社会意义。
以大鼠成骨样细胞ROS17/2.8为细胞模型,通过MTT法、ALP活性检测、ALP染色、通过流式细胞仪测定成骨样细胞钙离子摄取,及用茜素红S法来对矿化结节计数观察对塞隆骨提取物成骨样细胞矿化功能的影响,在细胞水平上分析塞隆骨不同的提取组分对成骨样细胞ROS17/2.8增殖、分化、矿化的效果,并以激素类药物地塞米松作阳性对照。结果表明:塞隆骨脂提取物1×10~(-2)(g/ml)组、塞隆骨水煎液1×10~(-2)(g/ml)组、塞隆骨骨胶1×10~(-2)(g/ml)、1×10~(-2)(g/ml)组对成骨样细胞的增殖均较阳性、空白对照组强,且显著强于空白对照组
Myospalaxbaileyi, which belongs to Cricetidae family, Myospala subfamily and genera, is an underground rodent imperiling pasture and farmland on Qinghai-Tibet Plateau. It is one of the most devastating rodents for the Qinghai-Tibet Plateau grassland ecosystem. Because of its feeding and digging activity, every year it destroys millions of hektares of area and causes heavy economic loss to the Qinghai-Tibet Plateau.Sailonggu is the whole dehydrated skeleton of Myospalaxbaileyi and it' s the first-grade new medical material of Chinese traditional medicine authorized by the Ministry of Health in 1990. Researches have revealed that its pharmacal effect has similarities with tiger bones. Sailonggu can help relieve rheumatic pains, dispel pathogenic cold, remove dampness, induce menstruation, alleviate pain, and help build the liver and kidney. It' s also effective in the treatment of arthralgia, turgor, skin numbness, lassitude of waist and knee due to chill and damp arthralgia. Consequently it is an important component of many finished product of Chinese traditional medicine such as "Sailong wine for rheumatism" , "Saixue capsule for rheumatism" , "Jingzhu capsule for rheumatism" ,etc. Yet the research into its medical merit and action mechanism is not deep enough to study the concrete effective components.
Investigation into its action mechanism, quantity-effectiveness and time-effectiveness correlation and medical kinetics is deficient. Moreover, there is no report existing about its toxicity and sideeffect. This article uses Myospalaxbaileyi as the research target. Extract of Sailonggu from lipid, ethanol and water, and bone glue are made into watery decoction to probe into its medical merit; and by hydrolyzing Myospalaxbaileyi muscles with biological enzymes, we could expect overall development and utilization of Myospalaxbaileyi, improving it' s economic value and social significance.The experiment uses rat osteoblast ROS17/2.8 as the cell model, measures its calcium ion intake via MTT method, ALP activity detection and flow cytometry, observes the influence on its mineralization function by measuring the number of mineralization knots with alizarin red S method, analyses effectiveness of different Sailonggu extracts for the proliferation, differentiation and mineralization of osteoblast ROS17/2.8 on the cell level. The experiment also uses hormones medicine dexamethasone as a positive comparison. The outcome of the experiment proves that Sailonggu lipid extract 1×10-2 (g/ml) group, Sailonggu watery decoction 1×10-2 (g/ml) group, Sailonggu bone glue 1×10-1 (g/ml) and 1 ×10-2 (g/ml) group have more significant promotion to osteoblast' s proliferation when compared with blank comparison group and positive comparison group(dexamethasone group), with difference presenting significance value(p<0.05) if compared with the blank comparison group. When comparing the ALP staining result with the blank comparison group, it is shown that osteoblast cultured in those Sailonggu extracts are heavier stained by alkaline phosphatase. Positive cytoplasm stained by alkaline phosphatase should present light grey, sallow and grey black particles, the darker colour the higher content of the enzyme. Quantitative measurement of ALP indicates that after cultivation for 3 days, Sailonggu bone glue 1×10-1 (g/ml) and lipid extract 1 × 10-2 (g/ml)
groups have an obvious proliferation promotion (P<0.05). Those Sailonggu extract groups have stronger effect than the dexamethasone group and the treated cells have an apparently higher ALP level than the comparison group. The result indicates that Sailonggu extracts have an proliferation promotion to the R0S17/2. 8 rat osteoblast cultured in vitro; copared with blank comparison group, groups treated by medicine and positive comparison group obviously have more calcium knots (P <0.05) ; the medicine-treated groups and dexamethasone positive comparison group all have quite high calcium ingestion and 1×10-2(g/ml) bone glue group and lipid extract group showed quite high value which is significant compared with blank comparison group (P<0.05) . Sailonggu bone glue and lipid extract have a direct promotion for the mineralization of rat osteoblast cultured in vitro, and can improve osteoblast' s ability to form bones.The quantity and composition analysis of Myospalaxbaileyi flesh proved that its flesh has abundant protein, amino acid, unsaturated fatty acid, vitamine A, E, C,K and many kinds of microelement including Ca, Fe, K, Mn, Na and so on. The unsaturated fatty acid content is as high as 64. 14%, and linoleic acid(18:2) content is 28. 14%, higher than in any other animal fat. The EAA/TAA ratio reaches 39.65 % , close to FA0/WH0(1973) grading mode. The microelement content is characteristic in its high K and low Na content, according with the tendency of modern nourishment and health care.The optimum condition to decompose the flesh of Myospalaxbaileyi with papain is at the temperature of 55℃ for 6 hours, with the enzyme quantity at 2. 8%, solid-liquid ratio at 1:3.5 and pH at 6.0. Under this condition the hydrolysis extent of the flesh exceeds 48% and we could obtain 17 categories of amino acid including 8 essential ones that account for 46.58% of all the amino acid and have a rational proportion according with the human essential amino acid mode suggested by FAO/WHO, with an E/N value of 0. 92, larger than 0. 6 suggested by FAO/WHO. Because of the
high content of flavour amino acid, which occupies 26. 11% of the total amino acid, the digest is very delicious. In oder to probe into the physiological activity funtion of the digest, we employ the MTT method and neutral red colorimetric method to measure the effect it has on the proliferation of normal rat splenocyte and lymphocyte, phagocytic function of celiac macrophage and reticuloendothelial system, killing and toxicity property of NK cells, and the number of rat antibody-making splenocyte (IgM-PFC). Immunocompetence research on the zymolyte reveals that high and middle dosage (0. 4g/kg; 0. 2g/kg) have significant difference from the comparison group, especially the activity of the high dosage group. The zymolyte has a significant promotion on the cell and humoral immunity of normal rat as well as the killing activity and toxicity of NK cells.So as to further investigate the mechanism of Sailonggu' s promotion on osteoblast' s proliferation, differentiation and maturation, an osteoblast apoptosis experiment is designed, applying agarose gel electrophoresis and flow cytometer to study apoptosis of osteoblast. The result shows that osteoblast in Sailonggu extracts shows much fewer hypodiploid peeks. Agarose gel electrophoresis experiment proves that cells in blank comparison group present clear DNA Ladders whereas the Sailonggu ectract groups show no ladder-shaped DNA band. That means Sailonggu extracts can depress the apoptosis ratio of osteoblast and can postone its occurrence. The total RNA of osteoblast from the medicine-treated group and blank group are extracted to semi-quantif icationally determine the expression of IL-1 mRNA, IL-2 mRNA, IL-4 mRNA, IL-6 mRNA, IL-10 mRNA and IFN-γ mRNA with highly sensitive RT-PCR method. The extracts can promote the expression of a great variety of cell factor mRNA. As a whole, lipid extract and bone glue extract have stronger effect than dexamethasone and watery extract. These extracts could be effective in the bone metabolism through influencing the
expression of many cell factor genes.To find in the bone tissue the differently expressed genes related to curative effect of Sailonggu on osteoporosis after menopause, further illustrate the mechanism Chinese traditional medicine can prevent and cure osteoporosis, and to screen effective components in Sailonggu, we observe the influence of Sailonggu on the expression of OPG, OCN and cbfa genes in rat osteoblast cultured in vitro, exract the total mRNA in cells cultured for 3 days, and analyse the mRNA expression of OPG, OCN and cbfa using RT-PCR technique to make a semi-quantitative comparison. On the third day, the 3 genes all have some expression; relative to the blank comparison group, the Sailonggu extracts group and dexamethasone group increase the expression of OCN mRNA with extreme significance (P<0. 01), with Sailonggu extracts group having weaker effect than the dexamethasone group. Sailonggu extract groups, including lipid extract group 1×10-2, watery extract group 1×10-2 and bone glue group 1×10-3 , as well as dexamethasone group, all increase the expression of OPG mRNA(P<0. 01) while the dexamethasone group having stronger effect. Sailonggu watery extract group 1×10-2 and bone glue group 1×10-1 and dexamethasone group all increase the expression of OPG mRNA (P<0.05) with the lipid extract group 1×10-2 having stronger promotion than the dexamethasone group significantly (P<0. 01) .In order to study the anti-inflammation mechanism of different Sailonggu extracts, we do experiments with medicine filtration models which act on inflammation-related cell factors simultaneously. We adopt genetic transfection technique to obtain HepG2 cell line that can stably express recombinant protein, apply LPS to stimulate the formation of inflammation factors, qualitatively and quantitatively detect the fluorescence strength variation after treating the cell line with different extracts. And in these ways we probe into the feature of NF-kb activation caused by different Sailonggu extracts. Gelatingases in the
culture medium supernatant secreted by tumour cells and their active forms could be separated according to their different molecular weight by SDS-PAGE. SDS could be removed from protein by TritonX-100 so gelatingase can resume it' s protein degrading activity, which is used to evaluate the medicine' s ability to resist affection. We apply chemiluminescent COX inhibitor screening assay to detect influence on COX activity by different extracts to evaluate the medicine' s ability to resist affection and it turns out thai different Sailonggu extracts are capable of decreasing many inflammation factors with the lipid extraction having the best effect.To sum up, Sailonggu could promote proliferation and differentiation of rat osteoblast, the expression of OPG, OCN and cbfa mRNA, adjust the balance between osteoblast and osteoclast; and in these ways it can reconstruct the bones to prevent and cure osteoporosis. So it is suggested that Sailonggu could be a substitute for hormones to treat osteoporosis and has a great prospect to be developed into a new natural medical product to resist osteoporosis or bone absorption. Those Sailonggu extracts could be used as an antiinflammation Chinese traditional medicine, with the possibility to replace the tiger bone. And the zymolyte of Myospalaxbaileyi flesh has immunologic competence.
引文
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