目的:观察MAPK4基因敲除(KO)对小鼠脾脏免疫细胞组成的影响并探讨其意义。方法:利用基因同源重组对小鼠MAPK4基因进行敲除,PCR法鉴定MAPK4^-/-小鼠;Western blot检测小鼠脾脏中MAPK4蛋白表达;计算MAPK4^-/-小鼠脾脏的脏器指数和总细胞数...目的:观察MAPK4基因敲除(KO)对小鼠脾脏免疫细胞组成的影响并探讨其意义。方法:利用基因同源重组对小鼠MAPK4基因进行敲除,PCR法鉴定MAPK4^-/-小鼠;Western blot检测小鼠脾脏中MAPK4蛋白表达;计算MAPK4^-/-小鼠脾脏的脏器指数和总细胞数;HE染色观察脾脏的病理学变化;流式细胞术(FCM)分析小鼠脾脏中CD19^+B细胞和CD3^+T细胞的比例并计算细胞绝对数,并检测CD4^+T和CD8^+T细胞的比例及其活化相关膜分子CD62L和CD69的表达;最后,FCM分析脾脏中固有免疫细胞DC细胞、NK细胞、巨噬细胞和γδT细胞的比例并计算细胞绝对数。结果:与野生型(WT)小鼠相比,MAPK4^-/-小鼠的MAPK4蛋白表达水平明显降低( P <0.05);脏器指数无明显改变( P >0.05),脾脏细胞总数明显降低( P <0.05),脾小结增大,结构紊乱( P <0.05);CD19^+B细胞和CD3 ^+T细胞的细胞绝对数都明显降低( P <0.001);CD4 +T细胞的比例和绝对数均明显减少( P <0.01),同时,CD8 +T细胞的绝对数明显减少( P <0.001);固有免疫细胞中DC细胞、NK细胞、巨噬细胞和γδT细胞的细胞绝对数均明显降低( P <0.001)。结论: MAPK4敲除明显影响小鼠脾脏中免疫细胞的组成,提示其在机体免疫细胞发育和功能中具有重要作用。展开更多
Aralia taibaiensi,widely distributed in western China,particularly in the Qinba Mountains,has been utilized as a folk medicine for treating diabetes,gastropathy,rheumatism,and cardiovascular diseases.Saponins from A.t...Aralia taibaiensi,widely distributed in western China,particularly in the Qinba Mountains,has been utilized as a folk medicine for treating diabetes,gastropathy,rheumatism,and cardiovascular diseases.Saponins from A.taibaiensis(sAT)have demonstrated protective effects against oxidative stress and mitochondrial dysfunction induced by ischemia/reperfusion(I/R).However,the underlying mechanisms remain unclear.In vivo,middle cerebral artery occlusion/reperfusion(MCAO/R)induced inflammatory infiltration,neuronal injury,cell apoptosis,mitochondrial dysfunction,and oxidative stress in the ischaemic penumbra,which were effectively mitigated by sAT.sAT increased the mRNA and protein expression levels of apelin and its receptor apelin/apelin receptors(ARs)both in vivo and in vitro.(Ala13)-Apelin-13(F13A)and small interfering RNA(siRNA)abolished the regulatory effects of sAT on neuroprotection mediated by adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)/protein kinase B(Akt).Furthermore,sAT induced apelin/AR expression by simultaneously inhibiting P38 mitogen-activated protein kinase(P38 MAPK)/activating transcription factor 4(ATF4)and upregulating hypoxia-inducible factor-1α(HIF-1α).Our findings indicate that sAT regulates apelin/AR/AMPK by inhibiting P38 MAPK/ATF4 and upregulating HIF-1a,thereby suppressing oxidative stress and mitochondrial dysfunction.展开更多
AIM:To investigate the effects of a Chinese medicine formula“Qingxuan Runmu Yin”(QRY)on ocular surface inflammation in a rat model of dry eye,and its mechanism via the toll-like receptor 4(TLR4)/transforming growth ...AIM:To investigate the effects of a Chinese medicine formula“Qingxuan Runmu Yin”(QRY)on ocular surface inflammation in a rat model of dry eye,and its mechanism via the toll-like receptor 4(TLR4)/transforming growth factor kinase 1(TAK1)/p38 mitogen-activated protein kinase(p38MAPK)signaling pathway.METHODS:Seventy-two Sprague-Dawley rats were randomly divided into six groups(n=12 each):the control group,model group,3 groups of QRY(with low-,medium-,and high-doses),and SB203580 group.Dry eye was induced using benzalkonium chloride.Schirmer’s test(SIT)and corneal fluorescein staining(CFS)were performed every 14d throughout the experiment.Histopathological changes in corneal and conjunctival tissues were observed using hematoxylin and eosin(HE)and periodic acid-Schiff(PAS)staining.Protein expression levels of key inflammatory markers and signaling pathway targets were assessed via immunohistochemistry,ELISA,and Western blotting.RESULTS:Compared to the control group,the model group showed significant reductions in SIT and increases in CFS scores,alongside structural disorganization of corneal/conjunctival tissues,decreased conjunctival goblet cell(CGC)numbers,and elevated expression of inflammatory markers[interleukin(IL)-1β,IL-6,tumor necrosis factoralpha(TNF-α),matrix metalloproteinase-9(MMP9)]and pathway proteins(TLR4,p-TAK1,p-p38MAPK;P<0.05).Treatment with QRY(low,medium,and high doses)and SB203580 significantly improved SIT scores,reduced CFS scores,restored corneoconjunctival structure,increased CGC numbers,and decreased expression levels of IL-1β,IL-6,TNF-α,MMP9,TLR4,p-TAK1,and p-p38MAPK proteins compared to the model group(P<0.05).CONCLUSION:QRY may alleviate ocular surface inflammation associated with dry eye by inhibiting the TLR4/TAK1/p38MAPK signaling pathway,highlighting its potential therapeutic efficacy for dry eye.展开更多
目的基于网络药理学与分子对接技术,预测桔梗汤干预慢性咽炎的关键靶点及作用通路,并通过动物实验验证预测结果。方法建立乙型溶血性链球菌诱导的慢性咽炎大鼠模型,探究桔梗汤对慢性咽炎大鼠血清炎症因子及咽部组织病理变化的影响。使...目的基于网络药理学与分子对接技术,预测桔梗汤干预慢性咽炎的关键靶点及作用通路,并通过动物实验验证预测结果。方法建立乙型溶血性链球菌诱导的慢性咽炎大鼠模型,探究桔梗汤对慢性咽炎大鼠血清炎症因子及咽部组织病理变化的影响。使用人类在线孟德尔遗传数据库(Online Mendelian Inheritance in Man,OMIM)、DrugBank和GeneCards数据库筛选慢性咽炎关键靶点;UHPLC-QE-MS分析桔梗汤化学成分,结合中药系统药理学数据库与分析平台(Traditional Chinese Medicine Database and Analysis Platform,TCMSP)和SwisstargetPredicition数据库确认成分作用靶点。使用STRING平台构建关键靶点蛋白互作网络;通过注释、可视化和综合发现数据库(Database for Annotation,Visualization and Integrated Discovery,DAVID)对关键靶点进行基因本体(Gene Ontology,GO)分析和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)信号通路富集分析;通过AutoDock Tools 1.5.6软件对有效成分及核心靶点进行分子对接;Western Blot法验证预测结果。结果体内实验显示,桔梗汤可有效降低慢性咽炎大鼠血清中IL-1β、IL-6、TNF-α、ICAM-1、MCP1水平(P<0.05),并逆转咽部组织病理损伤。得到94个桔梗汤主要活性成分,协同作用于311个靶点,参与外源性刺激反应、药物反应、蛋白酪氨酸激酶活性等生物学过程,调控PI3K/AKT、MAPK、Ras等信号通路;分子对接结果显示关键靶点与有效成分间均有较强的结合能力;Western Blot显示桔梗汤可显著降低TLR4蛋白表达及P38 MAPK磷酸化水平。结论桔梗汤通过调控TLR4/P38 MAPK信号通路降低血清炎症因子及逆转咽部组织病理变化从而治疗慢性咽炎。展开更多
文摘目的:观察MAPK4基因敲除(KO)对小鼠脾脏免疫细胞组成的影响并探讨其意义。方法:利用基因同源重组对小鼠MAPK4基因进行敲除,PCR法鉴定MAPK4^-/-小鼠;Western blot检测小鼠脾脏中MAPK4蛋白表达;计算MAPK4^-/-小鼠脾脏的脏器指数和总细胞数;HE染色观察脾脏的病理学变化;流式细胞术(FCM)分析小鼠脾脏中CD19^+B细胞和CD3^+T细胞的比例并计算细胞绝对数,并检测CD4^+T和CD8^+T细胞的比例及其活化相关膜分子CD62L和CD69的表达;最后,FCM分析脾脏中固有免疫细胞DC细胞、NK细胞、巨噬细胞和γδT细胞的比例并计算细胞绝对数。结果:与野生型(WT)小鼠相比,MAPK4^-/-小鼠的MAPK4蛋白表达水平明显降低( P <0.05);脏器指数无明显改变( P >0.05),脾脏细胞总数明显降低( P <0.05),脾小结增大,结构紊乱( P <0.05);CD19^+B细胞和CD3 ^+T细胞的细胞绝对数都明显降低( P <0.001);CD4 +T细胞的比例和绝对数均明显减少( P <0.01),同时,CD8 +T细胞的绝对数明显减少( P <0.001);固有免疫细胞中DC细胞、NK细胞、巨噬细胞和γδT细胞的细胞绝对数均明显降低( P <0.001)。结论: MAPK4敲除明显影响小鼠脾脏中免疫细胞的组成,提示其在机体免疫细胞发育和功能中具有重要作用。
基金supported by the National Science Foundation of China(No.81903832),the Key Research and Development Plan of Shaanxi Province(No.2022SF-182),the Fundamental Research Funds for the Central Universities(No.D5000210799)Shaanxi Provincial Traditional Chinese Medicine Administration"Double Chain Integration"Middle Youth Research and Innovation Team Project(No.2022-SLRH-LJ-007)Shanghai Science and Technology Innovation Action Plan(No.22S21902600).
文摘Aralia taibaiensi,widely distributed in western China,particularly in the Qinba Mountains,has been utilized as a folk medicine for treating diabetes,gastropathy,rheumatism,and cardiovascular diseases.Saponins from A.taibaiensis(sAT)have demonstrated protective effects against oxidative stress and mitochondrial dysfunction induced by ischemia/reperfusion(I/R).However,the underlying mechanisms remain unclear.In vivo,middle cerebral artery occlusion/reperfusion(MCAO/R)induced inflammatory infiltration,neuronal injury,cell apoptosis,mitochondrial dysfunction,and oxidative stress in the ischaemic penumbra,which were effectively mitigated by sAT.sAT increased the mRNA and protein expression levels of apelin and its receptor apelin/apelin receptors(ARs)both in vivo and in vitro.(Ala13)-Apelin-13(F13A)and small interfering RNA(siRNA)abolished the regulatory effects of sAT on neuroprotection mediated by adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)/protein kinase B(Akt).Furthermore,sAT induced apelin/AR expression by simultaneously inhibiting P38 mitogen-activated protein kinase(P38 MAPK)/activating transcription factor 4(ATF4)and upregulating hypoxia-inducible factor-1α(HIF-1α).Our findings indicate that sAT regulates apelin/AR/AMPK by inhibiting P38 MAPK/ATF4 and upregulating HIF-1a,thereby suppressing oxidative stress and mitochondrial dysfunction.
基金Supported by National Natural Science Foundation of China(No.81973908)Natural Science Foundation of Heilongjiang Province(No.PL2024H224)Postgraduate Innovation Fund of Heilongjiang University of Chinese Medicine(No.2023yjscx018).
文摘AIM:To investigate the effects of a Chinese medicine formula“Qingxuan Runmu Yin”(QRY)on ocular surface inflammation in a rat model of dry eye,and its mechanism via the toll-like receptor 4(TLR4)/transforming growth factor kinase 1(TAK1)/p38 mitogen-activated protein kinase(p38MAPK)signaling pathway.METHODS:Seventy-two Sprague-Dawley rats were randomly divided into six groups(n=12 each):the control group,model group,3 groups of QRY(with low-,medium-,and high-doses),and SB203580 group.Dry eye was induced using benzalkonium chloride.Schirmer’s test(SIT)and corneal fluorescein staining(CFS)were performed every 14d throughout the experiment.Histopathological changes in corneal and conjunctival tissues were observed using hematoxylin and eosin(HE)and periodic acid-Schiff(PAS)staining.Protein expression levels of key inflammatory markers and signaling pathway targets were assessed via immunohistochemistry,ELISA,and Western blotting.RESULTS:Compared to the control group,the model group showed significant reductions in SIT and increases in CFS scores,alongside structural disorganization of corneal/conjunctival tissues,decreased conjunctival goblet cell(CGC)numbers,and elevated expression of inflammatory markers[interleukin(IL)-1β,IL-6,tumor necrosis factoralpha(TNF-α),matrix metalloproteinase-9(MMP9)]and pathway proteins(TLR4,p-TAK1,p-p38MAPK;P<0.05).Treatment with QRY(low,medium,and high doses)and SB203580 significantly improved SIT scores,reduced CFS scores,restored corneoconjunctival structure,increased CGC numbers,and decreased expression levels of IL-1β,IL-6,TNF-α,MMP9,TLR4,p-TAK1,and p-p38MAPK proteins compared to the model group(P<0.05).CONCLUSION:QRY may alleviate ocular surface inflammation associated with dry eye by inhibiting the TLR4/TAK1/p38MAPK signaling pathway,highlighting its potential therapeutic efficacy for dry eye.
文摘目的基于网络药理学与分子对接技术,预测桔梗汤干预慢性咽炎的关键靶点及作用通路,并通过动物实验验证预测结果。方法建立乙型溶血性链球菌诱导的慢性咽炎大鼠模型,探究桔梗汤对慢性咽炎大鼠血清炎症因子及咽部组织病理变化的影响。使用人类在线孟德尔遗传数据库(Online Mendelian Inheritance in Man,OMIM)、DrugBank和GeneCards数据库筛选慢性咽炎关键靶点;UHPLC-QE-MS分析桔梗汤化学成分,结合中药系统药理学数据库与分析平台(Traditional Chinese Medicine Database and Analysis Platform,TCMSP)和SwisstargetPredicition数据库确认成分作用靶点。使用STRING平台构建关键靶点蛋白互作网络;通过注释、可视化和综合发现数据库(Database for Annotation,Visualization and Integrated Discovery,DAVID)对关键靶点进行基因本体(Gene Ontology,GO)分析和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)信号通路富集分析;通过AutoDock Tools 1.5.6软件对有效成分及核心靶点进行分子对接;Western Blot法验证预测结果。结果体内实验显示,桔梗汤可有效降低慢性咽炎大鼠血清中IL-1β、IL-6、TNF-α、ICAM-1、MCP1水平(P<0.05),并逆转咽部组织病理损伤。得到94个桔梗汤主要活性成分,协同作用于311个靶点,参与外源性刺激反应、药物反应、蛋白酪氨酸激酶活性等生物学过程,调控PI3K/AKT、MAPK、Ras等信号通路;分子对接结果显示关键靶点与有效成分间均有较强的结合能力;Western Blot显示桔梗汤可显著降低TLR4蛋白表达及P38 MAPK磷酸化水平。结论桔梗汤通过调控TLR4/P38 MAPK信号通路降低血清炎症因子及逆转咽部组织病理变化从而治疗慢性咽炎。
