目的通过网络药理学和体内外实验研究,探讨铁死亡在龟鹿二仙胶治疗少弱精子症中的作用。方法通过TCMSP数据库、HERB数据库、PubChem数据库和Swiss数据库获取龟鹿二仙胶的活性成分和作用靶点,Genecard数据库和FerrDb数据库检索铁死亡基因...目的通过网络药理学和体内外实验研究,探讨铁死亡在龟鹿二仙胶治疗少弱精子症中的作用。方法通过TCMSP数据库、HERB数据库、PubChem数据库和Swiss数据库获取龟鹿二仙胶的活性成分和作用靶点,Genecard数据库和FerrDb数据库检索铁死亡基因,GEO数据库获取少弱精子症差异基因,三者取交集构建蛋白互作网络,进行基因本体(gene ontology,GO)功能和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析。使用H2O2刺激小鼠睾丸支持细胞构建氧化应激损伤模型,并用龟鹿二仙胶含药血清干预损伤模型细胞,观察细胞形态,检测细胞活性氧(reactive oxygen species,ROS)、乳酸脱氢酶(lactate dehydrogenase,LDH)、总铁和亚铁离子含量,透射电镜观察细胞超微结构,qRT-PCR和Western blotting检测细胞谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)和酰基辅酶A合成酶长链家族成员4(acyl-CoA synthetase long chain family member 4,ACSL4)的mRNA和蛋白表达。使用环磷酰胺构建小鼠少弱精子症模型,ELISA检测小鼠性激素水平,苏木素-伊红(HE)染色法观察小鼠睾丸组织病理学改变,免疫组化检测小鼠睾丸组织GPX4和ACSL4蛋白表达。结果共获取龟鹿二仙胶81个活性成分和190个作用靶点,686个铁死亡基因和4777个少弱精子症差异基因,龟鹿二仙胶干预少弱精子症铁死亡靶点共11个,GO富集分析共检测到869个条目,KEGG共有35条。体外实验显示,龟鹿二仙胶含药血清能显著降低细胞内ROS、LDH、总铁和亚铁离子含量(P<0.05、0.01),修复损伤线粒体,显著降低ACSL4的mRNA和蛋白表达(P<0.01),显著促进GPX4的mRNA和蛋白表达(P<0.01)。体内实验发现,龟鹿二仙胶能改善少弱精子症小鼠血清中性激素水平和睾丸组织病理损伤,显著降低小鼠睾丸组织中ACSL4蛋白表达(P<0.01),显著促进睾丸组织中GPX4蛋白表达(P<0.01)。结论龟鹿二仙胶能有效治疗少弱精子症,拮抗铁死亡的发生可能是龟鹿二仙胶治疗少弱精子症潜在的作用机制。展开更多
目的探究麻杏石甘汤通过外泌体miR-1249-5p靶向溶质载体家族4成员A1(solute carrier family 4 member 1,SLC4A1)抑制流感病毒诱导的肺上皮细胞损伤-巨噬细胞极化的级联损伤效应的作用机制。方法以肺上皮细胞为研究对象,采用CCK-8检测麻...目的探究麻杏石甘汤通过外泌体miR-1249-5p靶向溶质载体家族4成员A1(solute carrier family 4 member 1,SLC4A1)抑制流感病毒诱导的肺上皮细胞损伤-巨噬细胞极化的级联损伤效应的作用机制。方法以肺上皮细胞为研究对象,采用CCK-8检测麻杏石甘汤含药血清的安全剂量和对流感病毒刺激肺上皮细胞治疗剂量,NanoSight鉴定肺上皮细胞源外泌体的粒径和浓度,qRT-PCR检测外泌体中miR-1249-5p基因表达。以与肺上皮细胞源外泌体共培养的巨噬细胞为研究对象,采用qRT-PCR检测巨噬细胞中miR-1249-5p、SLC4A1和核因子-κB(nuclear factor-κB,NF-κB)通路相关基因表达;Western blotting检测巨噬细胞中SLC4A1和NF-κB通路相关蛋白表达;流式细胞术和qRT-PCR检测巨噬细胞M1/M2极化表型。结果CCK-8结果显示,10%和20%的麻杏石甘汤含药血清对肺上皮细胞无细胞毒性,显著抑制流感病毒诱导的肺上皮细胞损伤(P<0.05、0.01);NanoSight检测各组肺上皮细胞源外泌体大小无明显变化,外泌体浓度有所改变;qRT-PCR结果显示流感病毒刺激的肺上皮源外泌体miR-1249-5p基因表达明显降低(P<0.01),各给药组miR-1249-5p基因表达显著升高(P<0.05、0.001)。肺上皮源外泌体共培养后的巨噬细胞中miR-1249-5p基因表达与外泌体相一致(P<0.05、0.001);qRTPCR和Western blotting结果显示,模型组巨噬细胞SLC4A1和NF-κB通路基因和蛋白表达显著升高(P<0.01、0.001),各给药组SLC4A1和NF-κB通路基因和蛋白表达显著下降(P<0.05、0.01);流式细胞术结果显示模型组巨噬细胞CD86+比例明显增高(P<0.01),各给药组巨噬细胞CD86+比例显著降低(P<0.05);qRT-PCR结果显示共培养后模型组巨噬细胞肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)和IL-1β的表达显著升高(P<0.01、0.001),IL-10和转化生长因子-β1(transforming growth factor-β1,TGF-β1)的表达显著下降(P<0.05、0.01),各给药组以上因子表达趋势与之相反(P<0.05、0.01、0.001)。结论麻杏石甘汤可能通过肺上皮细胞源外泌体递送miR-1249-5p到巨噬细胞中,并靶向巨噬细胞中SLC4A1调控NF-κB信号通路,抑制流感病毒的细胞级联损伤效应的作用机制。展开更多
Background Uric acid （UA） is suspected to play a neuro-protective role in Parkinson＇s disease （PD）.This study aimed to evaluate whether the serum UA level was associated with the disease progression of PD in a re...Background Uric acid （UA） is suspected to play a neuro-protective role in Parkinson＇s disease （PD）.This study aimed to evaluate whether the serum UA level was associated with the disease progression of PD in a relatively large population of Chinese patients.Methods Serum UA levels were measured from 411 Chinese PD patients and 396 age-matched controls; following the uric acid colorimetric method,the serum creatinine （Scr） levels were also measured to reduce the bias caused by possible differences in renal excretion function.The disease progression was scored by Hoehn and Yahr （H＆Y） scales and disease durations; PD group was divided into 3 subgroups according to H＆Y scales.Independent-samples ttest was performed to analyze the differences between PD group and control group.Multiple analysis of covariance was performed to analyze the differences between PD subgroups.Spearman rank-correlation was performed to evaluate the associations between serum UA or Scr level and disease progression.Results PD patients were found to have significantly lower levels of serum UA than controls （（243.38±78.91） vs.（282.97±90.80） pmol/L,P〈0.01）.As the disease progression,the serum UA levels were gradually reduced.There was a significantly inverse correlation of UA levels with H＆Y scales （Rs=-0.429,P 〈0.01） and disease duration （Rs=-0.284,P 〈0.01） in PD patients of both females and males.No significant difference of the Scr level between PD patients and controls was found （（70.01±14.70） vs.（69.84±16.46） μmol/L）,and the Scr level was not involved in disease progression.Conclusion Lower serum UA levels may possess a higher risk of PD,which may be a potential useful biomarker to indicate the progression of PD.展开更多
文摘目的通过网络药理学和体内外实验研究,探讨铁死亡在龟鹿二仙胶治疗少弱精子症中的作用。方法通过TCMSP数据库、HERB数据库、PubChem数据库和Swiss数据库获取龟鹿二仙胶的活性成分和作用靶点,Genecard数据库和FerrDb数据库检索铁死亡基因,GEO数据库获取少弱精子症差异基因,三者取交集构建蛋白互作网络,进行基因本体(gene ontology,GO)功能和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析。使用H2O2刺激小鼠睾丸支持细胞构建氧化应激损伤模型,并用龟鹿二仙胶含药血清干预损伤模型细胞,观察细胞形态,检测细胞活性氧(reactive oxygen species,ROS)、乳酸脱氢酶(lactate dehydrogenase,LDH)、总铁和亚铁离子含量,透射电镜观察细胞超微结构,qRT-PCR和Western blotting检测细胞谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)和酰基辅酶A合成酶长链家族成员4(acyl-CoA synthetase long chain family member 4,ACSL4)的mRNA和蛋白表达。使用环磷酰胺构建小鼠少弱精子症模型,ELISA检测小鼠性激素水平,苏木素-伊红(HE)染色法观察小鼠睾丸组织病理学改变,免疫组化检测小鼠睾丸组织GPX4和ACSL4蛋白表达。结果共获取龟鹿二仙胶81个活性成分和190个作用靶点,686个铁死亡基因和4777个少弱精子症差异基因,龟鹿二仙胶干预少弱精子症铁死亡靶点共11个,GO富集分析共检测到869个条目,KEGG共有35条。体外实验显示,龟鹿二仙胶含药血清能显著降低细胞内ROS、LDH、总铁和亚铁离子含量(P<0.05、0.01),修复损伤线粒体,显著降低ACSL4的mRNA和蛋白表达(P<0.01),显著促进GPX4的mRNA和蛋白表达(P<0.01)。体内实验发现,龟鹿二仙胶能改善少弱精子症小鼠血清中性激素水平和睾丸组织病理损伤,显著降低小鼠睾丸组织中ACSL4蛋白表达(P<0.01),显著促进睾丸组织中GPX4蛋白表达(P<0.01)。结论龟鹿二仙胶能有效治疗少弱精子症,拮抗铁死亡的发生可能是龟鹿二仙胶治疗少弱精子症潜在的作用机制。
基金This work was supported by grants from the National High Technology Research and Development Program of China （No.2007AA022460）,the State Key Development Program for Basic Research of China （No.2011CB510000）,and the National Natural Science Foundation of China （No.81071032）.
文摘Background Uric acid （UA） is suspected to play a neuro-protective role in Parkinson＇s disease （PD）.This study aimed to evaluate whether the serum UA level was associated with the disease progression of PD in a relatively large population of Chinese patients.Methods Serum UA levels were measured from 411 Chinese PD patients and 396 age-matched controls; following the uric acid colorimetric method,the serum creatinine （Scr） levels were also measured to reduce the bias caused by possible differences in renal excretion function.The disease progression was scored by Hoehn and Yahr （H＆Y） scales and disease durations; PD group was divided into 3 subgroups according to H＆Y scales.Independent-samples ttest was performed to analyze the differences between PD group and control group.Multiple analysis of covariance was performed to analyze the differences between PD subgroups.Spearman rank-correlation was performed to evaluate the associations between serum UA or Scr level and disease progression.Results PD patients were found to have significantly lower levels of serum UA than controls （（243.38±78.91） vs.（282.97±90.80） pmol/L,P〈0.01）.As the disease progression,the serum UA levels were gradually reduced.There was a significantly inverse correlation of UA levels with H＆Y scales （Rs=-0.429,P 〈0.01） and disease duration （Rs=-0.284,P 〈0.01） in PD patients of both females and males.No significant difference of the Scr level between PD patients and controls was found （（70.01±14.70） vs.（69.84±16.46） μmol/L）,and the Scr level was not involved in disease progression.Conclusion Lower serum UA levels may possess a higher risk of PD,which may be a potential useful biomarker to indicate the progression of PD.
