Objective To establish an effective assay to access the effects of natural products on cathepsin K for screening antiosteoporosis drugs. Methods To obtain the purified cathepsin K, we cloned the target fragment from t...Objective To establish an effective assay to access the effects of natural products on cathepsin K for screening antiosteoporosis drugs. Methods To obtain the purified cathepsin K, we cloned the target fragment from the mRNA of human osteosacoma cell line MG63 and demonstrated its correctness through DNA sequencing. Cathepsin K was expressed in a high amount in E.coli after IPTG induction, and was purified to near homogenetity through resolution and column purification. The specificity of the protein was shown by Western blotting experiment. The biological activity of the components in the fermentation broth was assayed by their inhibitory effects on cathepsin K and its analog papain. Results With the inhibition of papain activity as a screen index, the fermentation samples of one thousand strains of fungi were tested and 9 strains among them showed strong inhibitory effects. The crude products of the fermentation broth were tested for their specific inhibitory effects on the purified human cathepsin K, the product of fungi 2358 shows the highest specificity against cathepsin K. Conclusions The compounds isolated from fungi 2358 show the highest biological activity and are worth further structure elucidation and function characterization.展开更多
Atherosclerosis is a chronic inflammatory disease, occurring preferentially in bifurcation, branching, and bending of blood vessels exposed to disturbed flow. Disturbed flow in atheroprone areas activates elevated pro...Atherosclerosis is a chronic inflammatory disease, occurring preferentially in bifurcation, branching, and bending of blood vessels exposed to disturbed flow. Disturbed flow in atheroprone areas activates elevated proteases, degrading elastin lamellae and collagenous matrix, resulting in endothelial dysfunction and vascular remodeling. As a mediator for extracellular matrix protein degradation, cathepsin K (CTSK) was directly regulated by hemodynamics and contributed to atherosclerosis. The mechanism of CTSK responding to disturbed flow and contributing to disturbed flow-induced atherosclerosis is unclear. In this study, the partial carotid ligation model of mice and in vitro disturbed shear stress model were constructed to explore the contribution and potential mechanism of CTSK in atherosclerosis. Our results indicated that CTSK elevated in the disturbed flow area in vivo and in vitro along with endothelial inflammation and atherogenesis. Additionally, the expression of integrin αvβ3 was upregulated in these atheroprone areas. We found that inhibition of the integrin αvβ3-cytoskeleton pathway could significantly block the activation of NF-κB and the expression of CTSK. Collectively, our findings unraveled that disturbed flow induces increased CTSK expression, and contributes to endothelial inflammation and vascular remodeling, leading to atherogenesis eventually. This study is helpful to provide new enlightenment for the therapy of atherosclerosis.展开更多
目的:探讨姜黄素抗C57BL/6小鼠肺纤维化分子机制。方法:博来霉素气管注射造小鼠肺纤维化模型,随机分组:对照组(气管内注入0.1mL生理盐水),模型组(气管内注入0.025U博来霉素),姜黄素组(气管内注入0.025U博来霉素);造模第2天,对照组和模...目的:探讨姜黄素抗C57BL/6小鼠肺纤维化分子机制。方法:博来霉素气管注射造小鼠肺纤维化模型,随机分组:对照组(气管内注入0.1mL生理盐水),模型组(气管内注入0.025U博来霉素),姜黄素组(气管内注入0.025U博来霉素);造模第2天,对照组和模型组给予生理盐水灌胃,姜黄素组给予200mg/kg/d姜黄素灌胃,分别于造模后第7、14、28天取材。采用Mallory染色观察肺组织胶原表达变化;应用免疫组化检测Cathepsin K蛋白的表达,经图像分析各组之间的差异;在免疫组化的基础上,Re-al Time RT-PCR,Western-blot进一步验证Cathepsin K mRNA及蛋白在各组之间表达的差异。结果:Mallory染色结果显示姜黄素组、模型组与对照组比较胶原表达都比对照组高,姜黄素组与模型组比较胶原表达减少;姜黄素组、模型组与对照组比较,Cathepsin K表达都有不同程度增加;但姜黄素组与模型组比较Cathepsin K蛋白表达显著增加,其中第14天(P=0.006);第7天(P=0.015);28天(P=0.027);Real Time RT-PCR分析CathepsinK mRNA表达,姜黄素组与模型组进行比较发现,姜黄素组比模型组明显增加,第7天(P=0.047),第14天(P=0.001),第28天增加不明显(P=0.053)。结论:姜黄素通过增加Cathepsin K的表达从而降解胶原而实现抗纤维化作用。展开更多
组织蛋白酶K(Cathepsin K,CTSK)属于半胱氨酸蛋白酶,是抗骨质疏松药物研发的重要靶点,在破骨细胞中特异性高表达,对降解骨胶原基质至关重要。与临床常用的抑制骨吸收药物相比,CTSK抑制剂具有在不影响骨形成的情况下有效减少骨吸收的优...组织蛋白酶K(Cathepsin K,CTSK)属于半胱氨酸蛋白酶,是抗骨质疏松药物研发的重要靶点,在破骨细胞中特异性高表达,对降解骨胶原基质至关重要。与临床常用的抑制骨吸收药物相比,CTSK抑制剂具有在不影响骨形成的情况下有效减少骨吸收的优点。迄今为止,CTSK抑制剂的研发均因安全性不足而失败,原因是对皮肤和血管系统等非骨组织造成副作用。传统中药富含抑制骨吸收的活性物质,一些文献表明中药来源的CTSK抑制剂抗骨质疏松作用明显,且没有明显的不良反应。笔者拟综述近10年中药来源的CTSK抑制剂的研究进展,资料主要来源于中国知网、万方和Web of Science数据库,为开发中药CTSK抑制剂和治疗CTSK相关疾病提供参考。展开更多
文摘Objective To establish an effective assay to access the effects of natural products on cathepsin K for screening antiosteoporosis drugs. Methods To obtain the purified cathepsin K, we cloned the target fragment from the mRNA of human osteosacoma cell line MG63 and demonstrated its correctness through DNA sequencing. Cathepsin K was expressed in a high amount in E.coli after IPTG induction, and was purified to near homogenetity through resolution and column purification. The specificity of the protein was shown by Western blotting experiment. The biological activity of the components in the fermentation broth was assayed by their inhibitory effects on cathepsin K and its analog papain. Results With the inhibition of papain activity as a screen index, the fermentation samples of one thousand strains of fungi were tested and 9 strains among them showed strong inhibitory effects. The crude products of the fermentation broth were tested for their specific inhibitory effects on the purified human cathepsin K, the product of fungi 2358 shows the highest specificity against cathepsin K. Conclusions The compounds isolated from fungi 2358 show the highest biological activity and are worth further structure elucidation and function characterization.
基金supported by The National Natural Science Foundation of China(No.11932014,32071312,31870939,31971239 and 12032007).
文摘Atherosclerosis is a chronic inflammatory disease, occurring preferentially in bifurcation, branching, and bending of blood vessels exposed to disturbed flow. Disturbed flow in atheroprone areas activates elevated proteases, degrading elastin lamellae and collagenous matrix, resulting in endothelial dysfunction and vascular remodeling. As a mediator for extracellular matrix protein degradation, cathepsin K (CTSK) was directly regulated by hemodynamics and contributed to atherosclerosis. The mechanism of CTSK responding to disturbed flow and contributing to disturbed flow-induced atherosclerosis is unclear. In this study, the partial carotid ligation model of mice and in vitro disturbed shear stress model were constructed to explore the contribution and potential mechanism of CTSK in atherosclerosis. Our results indicated that CTSK elevated in the disturbed flow area in vivo and in vitro along with endothelial inflammation and atherogenesis. Additionally, the expression of integrin αvβ3 was upregulated in these atheroprone areas. We found that inhibition of the integrin αvβ3-cytoskeleton pathway could significantly block the activation of NF-κB and the expression of CTSK. Collectively, our findings unraveled that disturbed flow induces increased CTSK expression, and contributes to endothelial inflammation and vascular remodeling, leading to atherogenesis eventually. This study is helpful to provide new enlightenment for the therapy of atherosclerosis.
文摘目的:探讨姜黄素抗C57BL/6小鼠肺纤维化分子机制。方法:博来霉素气管注射造小鼠肺纤维化模型,随机分组:对照组(气管内注入0.1mL生理盐水),模型组(气管内注入0.025U博来霉素),姜黄素组(气管内注入0.025U博来霉素);造模第2天,对照组和模型组给予生理盐水灌胃,姜黄素组给予200mg/kg/d姜黄素灌胃,分别于造模后第7、14、28天取材。采用Mallory染色观察肺组织胶原表达变化;应用免疫组化检测Cathepsin K蛋白的表达,经图像分析各组之间的差异;在免疫组化的基础上,Re-al Time RT-PCR,Western-blot进一步验证Cathepsin K mRNA及蛋白在各组之间表达的差异。结果:Mallory染色结果显示姜黄素组、模型组与对照组比较胶原表达都比对照组高,姜黄素组与模型组比较胶原表达减少;姜黄素组、模型组与对照组比较,Cathepsin K表达都有不同程度增加;但姜黄素组与模型组比较Cathepsin K蛋白表达显著增加,其中第14天(P=0.006);第7天(P=0.015);28天(P=0.027);Real Time RT-PCR分析CathepsinK mRNA表达,姜黄素组与模型组进行比较发现,姜黄素组比模型组明显增加,第7天(P=0.047),第14天(P=0.001),第28天增加不明显(P=0.053)。结论:姜黄素通过增加Cathepsin K的表达从而降解胶原而实现抗纤维化作用。
文摘组织蛋白酶K(Cathepsin K,CTSK)属于半胱氨酸蛋白酶,是抗骨质疏松药物研发的重要靶点,在破骨细胞中特异性高表达,对降解骨胶原基质至关重要。与临床常用的抑制骨吸收药物相比,CTSK抑制剂具有在不影响骨形成的情况下有效减少骨吸收的优点。迄今为止,CTSK抑制剂的研发均因安全性不足而失败,原因是对皮肤和血管系统等非骨组织造成副作用。传统中药富含抑制骨吸收的活性物质,一些文献表明中药来源的CTSK抑制剂抗骨质疏松作用明显,且没有明显的不良反应。笔者拟综述近10年中药来源的CTSK抑制剂的研究进展,资料主要来源于中国知网、万方和Web of Science数据库,为开发中药CTSK抑制剂和治疗CTSK相关疾病提供参考。
