BACKGROUND Calpain-2 is a Ca^2+-dependent cysteine protease,and high calpain-2 activity can enhance apoptosis mediated by multiple triggers.AIM To investigate whether calpain-2 can modulate aberrant endoplasmic reticu...BACKGROUND Calpain-2 is a Ca^2+-dependent cysteine protease,and high calpain-2 activity can enhance apoptosis mediated by multiple triggers.AIM To investigate whether calpain-2 can modulate aberrant endoplasmic reticulum(ER)stress-related apoptosis in rat hepatocyte BRL-3A cells.METHODS BRL-3A cells were treated with varying doses of dithiothreitol(DTT),and their viability and apoptosis were quantified by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-H-tetrazolium bromide and flow cytometry.The expression of ER stress-and apoptosis-related proteins was detected by Western blot analysis.The protease activity of calpain-2 was determined using a fluorescent substrate,Nsuccinyl-Leu-Leu-Val-Tyr-AMC.Intracellular Ca^2+content,and ER and calpain-2 co-localization were characterized by fluorescent microscopy.The impact of calpain-2 silencing by specific small interfering RNA on caspase-12 activation and apoptosis of BRL-3A cells was quantified.RESULTS DTT exhibited dose-dependent cytotoxicity against BRL-3A cells and treatment with 2 mmol/L DTT triggered BRL-3A cell apoptosis.DTT treatment significantly upregulated 78 kDa glucose-regulated protein,activating transcription factor 4,C/EBP-homologous protein expression by>2-fold,and enhanced PRKR-like ER kinase phosphorylation,caspase-12 and caspase-3 cleavage in BRL-3A cells in a trend of time-dependence.DTT treatment also significantly increased intracellular Ca^2+content,calpain-2 expression,and activity by>2-fold in BRL-3A cells.Furthermore,immunofluorescence revealed that DTT treatment promoted the ER accumulation of calpain-2.Moreover,calpain-2 silencing to decrease calpain-2 expression by 85%significantly mitigated DTT-enhanced calpain-2 expression,caspase-12 cleavage,and apoptosis in BRL-3A cells.CONCLUSION The data indicated that Ca^2+-dependent calpain-2 activity promoted the aberrant ER stress-related apoptosis of rat hepatocytes by activating caspase-12 in the ER.展开更多
目的:应用肌性斜颈兔动物模型,探讨针刀治疗肌性斜颈的作用机制及意义。方法:无水酒精注射制备动物肌性斜颈兔子模型后,选取模型兔子48只,随机分为正常组、模型组、针刀组、按摩组,每组12只。分别给予各组对应治疗,治疗2个月后各组分别...目的:应用肌性斜颈兔动物模型,探讨针刀治疗肌性斜颈的作用机制及意义。方法:无水酒精注射制备动物肌性斜颈兔子模型后,选取模型兔子48只,随机分为正常组、模型组、针刀组、按摩组,每组12只。分别给予各组对应治疗,治疗2个月后各组分别取部分胸锁乳突肌制作标本,免疫组化法观察钙蛋白酶1(calpain-1)、分化抗原决定簇45(CD45)、基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)、基质金属蛋白酶抑制因子2(tissue inhibitor of metalloproteinases-2,TIMP-2)在各组肌纤维中的表达情况。结果:与正常组比较,其余各组实验动物明显出现颈部活动受限、斜颈、头颈不对称症状,而治疗结束后,针刀组和按摩组实验动物颈部活动受限改善,无明显头颈不对称症状。calpain-1、CD45、TIMP-2的表达,与正常组比较,模型组、按摩组上调(P<0.01);与模型组比较,针刀组、按摩组下调(P<0.01);与针刀组比较,按摩组上调(P<0.01)。MMP-2表达,与正常组比较,模型组下调(P<0.01),与模型组比较,针刀组、按摩组上调(P<0.05)。结论:针刀治疗肌性斜颈效果显著,其作用机制可能与减少calpain-1、CD45、TIMP-2表达,增加MMP-2表达有关。展开更多
Virtual screening can be a helpful approach to propose treatments for COVID-19 by developing inhibitors for blocking the attachment of the virus to human cells. This study uses molecular docking, recovery time and dyn...Virtual screening can be a helpful approach to propose treatments for COVID-19 by developing inhibitors for blocking the attachment of the virus to human cells. This study uses molecular docking, recovery time and dynamics to analyze if potential inhibitors of main protease (M<sup>pro</sup>) of SARS-CoV-2 can interfere in the attachment of nanobodies, specifically Nb20, in the receptor binding domain (RBD) of SARS-CoV-2. The potential inhibitors are four compounds previously identified in a fluorescence resonance energy transfer (FRET)-based enzymatic assay for the SARS-CoV-2 M<sup>pro</sup>: Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376. The findings reveal that Boceprevir has the higher affinity with the RBD/Nb20 complex, followed by Calpain Inhibitor XII, GC376 and Calpain Inhibitor II. The recovery time indicates that the RBD/Nb20 complex needs a relatively short time to return to what it was before the presence of the ligands. For the RMSD the Boceprevir and Calpain Inhibitor II have the shortest interaction times, while Calpain Inhibitor XII shows slightly more interaction, but with significant pose fluctuations. On the other hand, GC376 remains stably bound for a longer duration compared to the other compounds, suggesting that they can potentially interfere with the neutralization process of Nb20.展开更多
基金the National Natural Science Foundation of China,No.81560105the Department of Science and Technology of Guizhou Province,No.LH(2014)7074。
文摘BACKGROUND Calpain-2 is a Ca^2+-dependent cysteine protease,and high calpain-2 activity can enhance apoptosis mediated by multiple triggers.AIM To investigate whether calpain-2 can modulate aberrant endoplasmic reticulum(ER)stress-related apoptosis in rat hepatocyte BRL-3A cells.METHODS BRL-3A cells were treated with varying doses of dithiothreitol(DTT),and their viability and apoptosis were quantified by 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-H-tetrazolium bromide and flow cytometry.The expression of ER stress-and apoptosis-related proteins was detected by Western blot analysis.The protease activity of calpain-2 was determined using a fluorescent substrate,Nsuccinyl-Leu-Leu-Val-Tyr-AMC.Intracellular Ca^2+content,and ER and calpain-2 co-localization were characterized by fluorescent microscopy.The impact of calpain-2 silencing by specific small interfering RNA on caspase-12 activation and apoptosis of BRL-3A cells was quantified.RESULTS DTT exhibited dose-dependent cytotoxicity against BRL-3A cells and treatment with 2 mmol/L DTT triggered BRL-3A cell apoptosis.DTT treatment significantly upregulated 78 kDa glucose-regulated protein,activating transcription factor 4,C/EBP-homologous protein expression by>2-fold,and enhanced PRKR-like ER kinase phosphorylation,caspase-12 and caspase-3 cleavage in BRL-3A cells in a trend of time-dependence.DTT treatment also significantly increased intracellular Ca^2+content,calpain-2 expression,and activity by>2-fold in BRL-3A cells.Furthermore,immunofluorescence revealed that DTT treatment promoted the ER accumulation of calpain-2.Moreover,calpain-2 silencing to decrease calpain-2 expression by 85%significantly mitigated DTT-enhanced calpain-2 expression,caspase-12 cleavage,and apoptosis in BRL-3A cells.CONCLUSION The data indicated that Ca^2+-dependent calpain-2 activity promoted the aberrant ER stress-related apoptosis of rat hepatocytes by activating caspase-12 in the ER.
文摘目的:应用肌性斜颈兔动物模型,探讨针刀治疗肌性斜颈的作用机制及意义。方法:无水酒精注射制备动物肌性斜颈兔子模型后,选取模型兔子48只,随机分为正常组、模型组、针刀组、按摩组,每组12只。分别给予各组对应治疗,治疗2个月后各组分别取部分胸锁乳突肌制作标本,免疫组化法观察钙蛋白酶1(calpain-1)、分化抗原决定簇45(CD45)、基质金属蛋白酶2(matrix metalloproteinase-2,MMP-2)、基质金属蛋白酶抑制因子2(tissue inhibitor of metalloproteinases-2,TIMP-2)在各组肌纤维中的表达情况。结果:与正常组比较,其余各组实验动物明显出现颈部活动受限、斜颈、头颈不对称症状,而治疗结束后,针刀组和按摩组实验动物颈部活动受限改善,无明显头颈不对称症状。calpain-1、CD45、TIMP-2的表达,与正常组比较,模型组、按摩组上调(P<0.01);与模型组比较,针刀组、按摩组下调(P<0.01);与针刀组比较,按摩组上调(P<0.01)。MMP-2表达,与正常组比较,模型组下调(P<0.01),与模型组比较,针刀组、按摩组上调(P<0.05)。结论:针刀治疗肌性斜颈效果显著,其作用机制可能与减少calpain-1、CD45、TIMP-2表达,增加MMP-2表达有关。
文摘Virtual screening can be a helpful approach to propose treatments for COVID-19 by developing inhibitors for blocking the attachment of the virus to human cells. This study uses molecular docking, recovery time and dynamics to analyze if potential inhibitors of main protease (M<sup>pro</sup>) of SARS-CoV-2 can interfere in the attachment of nanobodies, specifically Nb20, in the receptor binding domain (RBD) of SARS-CoV-2. The potential inhibitors are four compounds previously identified in a fluorescence resonance energy transfer (FRET)-based enzymatic assay for the SARS-CoV-2 M<sup>pro</sup>: Boceprevir, Calpain Inhibitor II, Calpain Inhibitor XII, and GC376. The findings reveal that Boceprevir has the higher affinity with the RBD/Nb20 complex, followed by Calpain Inhibitor XII, GC376 and Calpain Inhibitor II. The recovery time indicates that the RBD/Nb20 complex needs a relatively short time to return to what it was before the presence of the ligands. For the RMSD the Boceprevir and Calpain Inhibitor II have the shortest interaction times, while Calpain Inhibitor XII shows slightly more interaction, but with significant pose fluctuations. On the other hand, GC376 remains stably bound for a longer duration compared to the other compounds, suggesting that they can potentially interfere with the neutralization process of Nb20.
