豨莶草提取物对膝骨关节炎大鼠软骨组织损伤及NOX2/ROS/NF-κB通路的影响

目的观察豨莶草提取物对膝骨关节炎(KOA)大鼠软骨组织病理损伤的影响,并探讨其与NOX2/ROS/NF-κB通路的关系。方法将75只雄性SD大鼠随机分为假手术组、模型组和豨莶草低、中、高剂量组各15只。模型组和豨莶草不同剂量组向大鼠后肢双侧膝关节腔注射碘乙酸钠构建KOA模型;假手术组注射生理盐水。造模后,豨莶草低、中、高剂量组分别给予75、150、300 mg/kg豨莶草提取物经口灌服,假手术组和模型组给予生理盐水经口灌服。处死大鼠,取后肢双侧膝关节,用番红固绿法染色,采用Mankin评分评价关节软骨损伤情况;取后肢双侧膝关节软组织,采用硫代巴比妥酸法检测MDA含量,氮蓝四唑法检测SOD含量;RT-PCR法检测膝关节组织NOX2、NF-κB mRNA表达,Western blotting法检测膝关节组织NOX2、NF-κB蛋白表达。结果与假手术组相比,模型组Mankin评分升高,MDA含量升高,SOD含量降低,NOX2、NF-κB mRNA及蛋白表达均升高(P均<0.05);与模型组相比,豨莶草低、中、高剂量组Mankin评分、MDA含量、NOX2和NF-κB mRNA及蛋白表达均降低,中、高剂量组SOD含量升高(P均<0.05);与低剂量组相比,中、高剂量组Mankin评分、MDA含量、NOX2 mRNA和蛋白、NF-κB蛋白表达量及高剂量组NF-κB mRNA降低,高剂量组SOD含量升高(P均<0.05);与中剂量组相比,高剂量组Mankin评分、MDA含量、NOX2 mRNA和蛋白、NF-κB蛋白表达量降低,SOD含量升高(P均<0.05)。结论豨莶草提取物能够减轻KOA大鼠关节软骨组织的病理性损伤,其机制可能与调节NOX2/ROS/NF-κB通路有关。

紫云英苷通过NOX2/ROS/NF-κB信号通路抑制哮喘气道炎症

目的探究紫云英苷(astragalin,AG)对哮喘小鼠气道炎症的影响及作用机制。方法SPF级雄性小鼠50只随机分为正常组、哮喘模型组、紫云英苷低(AG25)、中(AG50)、高(AG100)剂量组。鸡卵白蛋白吸入制备哮喘小鼠模型,收取支气管肺泡灌洗液(BALF)后,Diff-Quik染色计数细胞数量,ELISA检测BALF中IL-4、IL-5、IL-13水平。HE染色观察小鼠肺组织炎症变化。利用DCFH-DA探针检测ROS水平,比色法检测SOD活性和MDA含量。Western blot检测肺组织IL-4、IL-5、IL-13、NOX2、p47phox、p-NF-κB p65、NF-κB p65、IκBα、p-IκBα和β-actin表达。结果AG明显减少BALF各炎症细胞及总细胞数量,降低BALF和肺组织IL-4、IL-5、IL-13含量,减少肺组织炎症细胞浸润。AG抑制NOX2和p47phox表达,降低ROS水平和MDA水平,提高SOD活性,抑制IκBα和NF-κB p65磷酸化。结论AG通过NOX2/ROS/NF-kB信号通路调节氧化应激反应减轻哮喘气道炎症。

Study on the Hepatoprotective Effect of Oxalis coriniculata L. and Related Mechanism by Regulating Oxidative Stress and TLR-2/NF-κB Signaling Pathway

[Objectives]This study aimed to explore the protective effect of Oxalis coriniculata L.on rats with acute liver injury induced by carbon tetrachloride(CCl4)and related mechanism by regulating oxidative stress and the TLR-2 TLR-2/NF-κB signaling pathway.[Methods]A total of 48 female rats were randomly and evenly divided into normal group,model group,silymarin group(0.12 g/kg),and high(16 g/kg),middle(8 g/kg)and low-dose(4 g/kg)O.coriniculata L.groups.The rats in the groups were intragastrically administered with 5 mL/kg of corresponding drugs(equal-volume distilled water for normal group and control group),respectively.The administration was conducted twice a day,for 10 consecutive days.After 2 h of the last administration,the rats in all the groups except the normal group were intraperitoneally injected with 12%carbon tetrachloride(CCl4)olive oil solution(5 mL/kg),respectively to establish liver injury rat models.After 16 h,the eyeball blood of the rats was collected,and their liver tissues were collected for preparation of HE sections.The biochemical indicators detected included aspartate aminotransferase(AST),alanine aminotransferase(ALT),total superoxide dismutase(T-SOD)and glutathione peroxidase(GSH-Px)activity and malondialdehyde(MDA)content in the serum.The contents of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in the serum were detected by ELISA.The expression of Toll-like receptor-2(TLR-2)and nuclear factor-κB(NF-κB)in liver tissue was detected using Western blotting.The pathological changes of liver were observed under light microscope.[Results]Compared with the normal group,the ALT,AST activity and MDA,IL-1β,IL-6,TNF-αlevels in rat serum significantly increased(P<0.01),the GSH-Px,T-SOD activity in rat serum significantly decreased(P<0.01),and the expression of TLR-2 and NF-κB in liver tissue was up-regulated(P<0.01)in the model group.Compared with the model group,the ALT,AST activity and MDA,IL-1β,IL-6 and TNF-αlevels in rat serum reduced(P<0.05,P<0.01),the GSH-Px and T-SOD activity in rat serum increased(P<0.05,P<0.01),and the expression of TLR-2 and NF-κB in liver tissue was down-regulated(P<0.05,P<0.01)in the O.coriniculata L.administration groups.Pathological sections show that O.coriniculata L.had an improving effect on rats with acute liver injury induced by CCl4.[Conclusions]O.coriniculata L.has a good protective effect on rats with acute liver injury induced by CCl4.Its mechanism may be related to inhibition of oxidative stress,inhibition of inflammatory response and regulation of the TLR-2/NF-κB signaling pathway.

Neuroprotective effect of Angiopep-2 peptide modified scutellarin-loaded PEGylated PAMAM dendrimer nanoparticles on ischemic stroke by modulating the Toll-like receptors-dependent MyD88/IKK/NF-κB signaling pathway

OBJECTIVE The greatest challenge in chemotherapy of ischemic stroke is the construction a suitable delivery system to overcome the poor physicochemical properties of drug and its low permeability across the blood brain barrier(BBB).METHODS In the present study,dendrimer,polyamidoamine(PAMAM),was synthesized as the nano-drug carriers.Angiopep-2,which has been proved excellent ability to cross the BBB,was exploited as the targeting ligand to conjugate PAMAM via bifunctional polyethylene glycol(PEG).Then scutellarin(STA)was encapsulated into the functionalized nanoparticles(NPs)to formulate Angiopep-2 modified STA-loaded PEG-PAMAM NPs.Ischemic stroke model was established to evaluate the treatment efficacy and protective mechanism of Angiopep-2-STA-PEG-PAMAM NPs.RESULTS The pharmacokinetics and biodistribu-tion demonstrated that Angiopep-2-STA-PEG-PAMAM NPs exhibited significantly higher plasma concentration from 1 h to 10 h after intravenous administration and improve accumulation in brain(4.7-fold)compared with STA solution.Moreover,prolonged elimination half-life(4.8-fold)and lower clearance(3.4-fold)were observed.The brain uptake study of 6-coumarin confirmed that Angiopep-2-PEG-PAMAM NPs possessed better brain targeting efficacy(3.2-fold)than PEG-PAMAM NPs.Angiopep-2-STA-PEG-PAMAM NPs obviously ameliorated infarct volume,neurological deficit,histopathological severity and neuronal apoptosis.In addition,Angiopep-2-STA-PEG-PAMAM NPs markedly inhibited the calcium content and the levels of IL-12p40,IL-13,IL-17 and IL-23.Furthermore,Angiopep-2-STA-PEG-PAMAM NPs significantly decreased the m RNA and protein expressions of HMGB1,TLR2,TLR4,TLR5,My D88,TRIF,TRAM,IRAK-4,TRAF6,IкBα,IKKβand NF-кBp65.CONCLUSION The results suggested that Angiopep-2modified scutellarin-loaded PEG-PAMAM nanocarriers possessed remarkable neuroprotective effects on ischemic stroke through modulation of inflammatory cascades and HMGB1/TLRs/MyD 88-induced NF-κB activation pathways.

健脾清化散瘀饮对慢性萎缩性胃炎模型大鼠胃黏膜TXNDC5、NOX2、NF-κB、TNF-α表达的影响

目的观察健脾清化散瘀饮对慢性萎缩性胃炎(CAG)大鼠胃黏膜炎症水平、萎缩程度,以及含硫氧还蛋白结构域5(TXNDC5)、吞噬细胞NADPH氧化酶(NOX2)、核因子κB(NF-κB)、肿瘤坏死因子-α(TNF-α)表达影响。方法 Wistar清洁级大鼠按照随机数字法分为空白对照组(n=8)与造模组(n=42),采用"氨水+去氧胆酸钠+乙醇法灌胃"法建立CAG大鼠模型,造模成功后,造模组随机分中药低、中、高治疗组,给予相应药物连续干预4周后,采用HE染色检测大鼠胃黏膜病变情况,采用免疫组化法检测胃组织中TXNDC5、NOX2、NF-κB、TNF-α蛋白表达。结果与空白对照组相比,模型组大鼠炎症细胞浸润明显,胃黏膜固有腺体萎缩、减少,胃黏膜组织中TXNDC5、NOX2、NF-κB、TNF-α蛋白表达显著升高(P<0.05或P<0.01)。与模型组比较,健脾清化散瘀饮低、中、高剂量治疗组胃黏膜固有腺体萎缩及炎症程度明显改善,组织中TXNDC5、NOX2、NF-κB、TNF-α蛋白表达水平均有所下降,差异具有统计学意义(P<0.05或P<0.01)。结论健脾清化散瘀饮可有效改善CAG大鼠胃黏膜组织病理改变,其机制可能与降低胃黏膜组织中TXNDC5、NOX2、NF-κB、TNF-α表达有关。

β-arrestin 2 attenuates lipopolysaccharide-induced liver injury via inhibition of TLR4/NF-κB signaling pathwaymediated inflammation in mice

AIM To study the role and the possible mechanism of β-arrestin 2 in lipopolysaccharide(LPS)-induced liver injury in vivo and in vitro.METHODS Male β-arrestin 2^(+/+) and β-arrestin 2^(-/-)C57 BL/6 J mice were used for in vivo experiments, and the mouse macrophage cell line RAW264.7 was used for in vitro experiments. The animal model was established via intraperitoneal injection of LPS or physiological sodium chloride solution. Blood samples and liver tissues were collected to analyze liver injury and levels of pro-inflammatory cytokines. Cultured cell extracts were collected to analyze the production of pro-inflammatory cytokines and expression of key molecules involved in the TLR4/NF-κB signaling pathway.RESULTS Compared with wild-type mice, the β-arrestin 2 knockout mice displayed more severe LPS-induced liver injury and significantly higher levels of proinflammatory cytokines, including interleukin(IL)-1β, IL-6, tumor necrosis factor(TNF)-α, and IL-10. Compared with the control group, pro-inflammatory cytokines(including IL-1β, IL-6, TNF-α, and IL-10) produced by RAW264.7 cells in the β-arrestin 2 si RNA group were significantly increased at 6 h after treatment with LPS. Further, key molecules involved in the TLR4/NF-κB signaling pathway, including phosphoIκBα and phosho-p65, were upregulated.CONCLUSION β-arrestin 2 can protect liver tissue from LPS-induced injury via inhibition of TLR4/NF-κB signaling pathwaymediated inflammation.

Non-SMC condensin Ⅰ complex subunit D2 and non-SMC condensin Ⅱ complex subunit D3 induces inflammation via the IKK/NF-κB pathway in ulcerative colitis

BACKGROUND Ulcerative colitis(UC)is a chronic,nonspecific intestinal inflammatory disease with undefined pathogenesis.Non-SMC condensin I complex subunit D2(NCAPD2)and non-SMC condensin II complex subunit D3(NCAPD3)play pivotal roles in chromosome assembly and segregation during both mitosis and meiosis.To date,there has been no relevant report about the functional role of NCAPD2 and NCAPD3 in UC.AIM To determine the level of NCAPD2/3 in intestinal mucosa and explore the mechanisms of NCAPD2/3 in UC.METHODS Levels of NCAPD2/3 in intestinal tissue were detected in 30 UC patients and 30 healthy individuals with in situ hybridization(ISH).In vitro,NCM60 cells were divided into the NC group,model group,si-NCAPD2 group,si-NCAPD3 group and si-NCAPD2+si-NCAPD3 group.Inflammatory cytokines were measured by ELISA,IKK and NF-κB were evaluated by western blot,and IKK nucleation and NF-κB volume were analyzed by immunofluorescence assay.RESULTS Compared with expression in healthy individuals,NCAPD2 and NCAPD3 expression in intestinal tissue was significantly upregulated(P<0.001)in UC patients.Compared with levels in the model group,IL-1β,IL-6 and TNF-αin the si-NCAPD2,si-NCAPD3 and si-NCAPD2+si-NCAPD3 groups were significantly downregulated(P<0.01).IKK and NF-κB protein expression in the si-NCAPD2,si-NCAPD3 and si-NCAPD2+si-NCAPD3 groups was significantly decreased(P<0.01).Moreover,IKK nucleation and NF-κB volume were suppressed upon si-NCAPD2,si-NCAPD3 and si-NCAPD2+si-NCAPD3 transfection.CONCLUSION NCAPD2/3 is highly expressed in the intestinal mucosa of patients with active UC.Overexpression of NCAPD2/3 promotes the release of pro-inflammatory cytokines by modulating the IKK/NF-κB signaling pathway.

Effects of Polysaccharides from Dicliptera chinensis(L.)Nees.on TLR4/NF-κB Signaling Pathway in Cell Model of Non-alcoholic Fatty Liver

[Objectives]To observe the effects of polysaccharides from Dicliptera chinensis(L.)Nees.on the expression of TLR/NF-κB pathway related proteins in HepG2 cells induced by oleic acid,and to explore the possible mechanism of polysaccharides from D.chinensis(L.)Nees.in the treatment of non-alcoholic fatty liver disease(NAFLD).[Methods]HepG2 cells were induced with oleic acid to establish a non-alcoholic fatty liver cell model.After intervention with 0.25 and 0.5 mg/mL of D.chinensis(L.)Nees.polysaccharides,the ALT and AST activity and TG and TC contents were detected with kits,and the changes in the expression of CDK5,TLR4,p-NF-κB and NF-κB were analyzed using Western-blotting.[Results]In the HepG2 cells induced with oleic acid,the ALT and AST activity increased significantly,the TG and TC contents increased significantly,and the expression levels of CDK5,TLR4 and p-NF-κB proteins up-regulated significantly.In the HepG2 cells intervened with D.chinensis(L.)Nees.polysaccharides,the activity of ALT and AST,the contents of TG and TC,and the expression levels of CDK5,TLR4 and p-NF-κB proteins all reduced significantly.[Conclusions]Polysaccharides from D.chinensis(L.)Nees.may interfere with NAFLD by inhibiting the TLR4/NF-κB pathway.

黑逍遥散调控NOX2/ROS/NF-κB信号通路干预AD模型小鼠小胶质细胞极化

基于氧化酶2(NADPH oxidase 2,NOX2)/活性氧簇(reactive oxygen species,ROS)/核转录因子κB(nuclear factor kappaB,NF-κB)信号通路探究黑逍遥散对APP/PS1双转基因小鼠小胶质细胞(microglia,MG)极化的影响及作用机制。4月龄雄性APP/PS1双转基因小鼠50只,随机分为模型组、MCC950组(10 mg·kg-1)及黑逍遥散低、中、高剂量组(6.45、12.89、25.78 g·kg-1),同月龄、同系种雄性C57BL/6J小鼠30只,随机分为空白组、空白灌胃干预组和空白腹腔注射组,各组给药干预90 d。Morris水迷宫检测学习认知能力,尼氏染色和透射电镜观察海马神经元病理形态和超微结构,免疫荧光检测小胶质细胞M1型标志物CD16/32+/Iba-1^(+)、M2型标志物CD206^(+)/Iba-1^(+)的阳性表达及海马ROS表达情况,比色法检测海马丙二醛(malondialdehyde,MDA)和超氧化物歧化酶(superoxide dismutase,SOD)含量,酶联免疫吸附法检测海马白细胞介素-6(interleukin-6,IL-6)、白细胞介素-8(interleukin-8,IL-8)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)炎症因子含量,蛋白免疫印迹检测海马β-淀粉样蛋白(β-amyloid protein,Aβ)、Iba-1、CD16/32、CD206、NOX2、NF-κB、p-NF-κB、核因子κB抑制蛋白α(NF-κB inhibitor alpha,IκBα)和p-IKBα蛋白表达。结果显示,与空白组比较,模型组小鼠目标象限运动距离、逃避潜伏期显著延长(P<0.01),目标象限滞留时间及百分比显著缩短(P<0.01);神经元细胞排列紊乱,出现肿胀、核消失或偏置,细胞数量明显减少,尼氏小体溶解甚至消失,胞质呈透亮区域;细胞膜出现缺损、皱缩,形态异常,胞质中少见细胞器,线粒体明显肿大,数量减少;小胶质细胞M1型标志物CD16/32+/Iba-1^(+)显著升高(P<0.01),M2型标志物CD206^(+)/Iba-1^(+)显著降低(P<0.01),ROS活性和MDA含量显著升高(P<0.01),SOD水平显著降低(P<0.01),炎症因子IL-6、IL-8、TNF-α含量显著升高(P<0.01);Aβ、CD16/32、Iba-1、NOX2、NF-κB和IKBα蛋白表达及磷酸化显著升高(P<0.01),CD206显著降低(P<0.01)。空白组与空白灌胃干预组、空白腹腔注射组差异无统计学意义。黑逍遥散干预后,各剂量组小鼠目标象限运动距离和逃避潜伏期显著缩短(P<0.01),目标象限滞留时间及百分比显著延长(P<0.01);细胞数量明显增多,排列较为整齐,肿胀明显缓解,尼氏小体增加;细胞形态较为规则,胞膜较为完整,线粒体肿胀明显缓解,但仍有部分内质网轻度扩张;M1型标志物CD16/32+/Iba-1^(+)显著降低(P<0.05或P<0.01),M2型标志物CD206^(+)/Iba-1^(+)显著升高(P<0.01),ROS活性、MDA含量显著降低(P<0.01),SOD水平显著升高(P<0.01),炎症因子IL-6、IL-8、TNF-α含量显著降低(P<0.01);Aβ、CD16/32、Iba-1、NOX2、NF-κB、IKBα蛋白及其磷酸化显著降低(P<0.01),CD206显著升高(P<0.01)。综上所述,黑逍遥散能够缓解神经元损伤,提高APP/PS1小鼠学习记忆能力,作用机制可能与抑制NOX2/ROS/NF-κB信号通路,调节MG极化,增加M2型表达,抑制M1型表达,减少炎症因子释放有关。

NF-κB在电刺激引起C2C12肌管线粒体功能低下时的作用

目的:探讨NF-κB在不同电刺激状态下C2C12肌管粒体功能低下时的作用。方法:采用TY-C型电刺激器(强度为45 V、20 ms、5 Hz)刺激分化6天的C2C12肌管来建立耐力运动时的神经冲动模型,对照组无任何电刺激,实验组分为刺激即刻组、孵育组和对照组,刺激即刻组的刺激时间分别为60分钟、75分钟、90分钟、120分钟、150分钟和180分钟,孵育组刺激120分钟后放入培养箱中继续培养,时间分别为30分钟、60分钟、120分钟和180分钟。测定各组细胞pIKK-α、NF-κB及MnSOD活性变化。结果:与对照组相比,电刺激即刻组C2C12细胞NF-κB活性显著升高(P<0.01),且刺激120分钟和150分钟增加明显(P<0.05),到180分钟时,略有下降。孵育组中孵育120分钟时NF-κB活性较孵育组其它细胞显著增加(P<0.01);与对照组相比,刺激即刻各组细胞内pIKK-α表达显著增加(P<0.05),但不同时间电刺激各组之间无显著差别。孵育组中,孵育120分钟细胞pIKK-α蛋白表达与孵育组其他细胞相比显著升高(P<0.01);与对照组相比,刺激即刻组刺激150分钟时细胞线粒体MnSOD活性显著增加(P<0.01),孵育组孵育120分钟时C2C12细胞线粒体MnSOD活性显著增加(P<0.01)。结论:(1)在电刺激引起C2C12细胞产生氧化应激,导致细胞线粒体功能的下降中,内源性ROS的产生会激活NF-κB的表达,同时激活IKK-α的磷酸化,且在电刺激强度一定时,NF-κB的活性对电刺激的应答有一定的时相性。(2)随着刺激时间的不同,细胞线粒体MnSOD的活性变化有差异,且线粒体MnSOD活性的变化与刺激时间有关,推测NF-κB的表达上调了细胞内抗氧化物酶MnSOD的表达,这在一定程度上对细胞起到保护作用。

高糖通过激活活性氧介导的NF-κB信号通路诱导HK-2人肾小管上皮细胞转分化

目的基于活性氧/核因子κB(ROS/NF-κB)信号通路探讨高糖诱导的人肾小管上皮细胞转分化机制。方法 HK-2正常人近端肾小管上皮细胞随机分为空白对照组、渗透压对照组、高糖组、吡咯烷二硫氨基甲酸(PDTC)组。用相差显微镜观察细胞形态、噻唑蓝(MTT)法检测细胞活性,流式细胞术检测细胞内ROS含量, ELISA检测丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性;Western blot法检测细胞NF-κBp65、磷酸化的NF-κB抑制蛋白α(p-IκBα)和IκB激酶α(IKKα)、单核细胞趋化蛋白1(MCP-1)和细胞间黏附分子1(ICAM-1)的蛋白水平;免疫细胞化学法检测细胞NF-κBp65、上皮型钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-SMA)表达。结果高糖组细胞变成长梭形或不规则形,边缘可呈现放射状,细胞间隙变大,折光度下降,排列不整齐。同时细胞活性随处理时间的延长不断下降, PDTC组细胞活性较高糖组高。与空白对照组相比,高糖组ROS及MDA含量增加、SOD活性下降, PDTC组ROS及MDA含量较高糖组减少、SOD活性较高糖组增强。与空白组比较,高糖组NF-κBp65、p-IκBα、IKKα、MCP-1、ICAM-1蛋白水平增加;与高糖组比较PDTC组以上蛋白水平降低。与空白组比较,高糖组NF-κBp65及α-SMA的阳性细胞所占比率升高、E-cadherin的阳性细胞所占比率降低;与高糖组比较, PDTC组NF-κBp65及α-SMA的阳性细胞所占比率降低、E-cadherin的阳性细胞比率升高。结论高糖可以诱导HK-2细胞发生上皮间质转化, ROS介导的NF-κB信号通路的激活参与以上进程, PDTC可阻断该过程。

Fucoxanthin suppresses OxLDL-induced inflammation via activation of Nrf2 and inhibition of NF-κB signaling

Objective:To explore the impact of fucoxanthin on oxidized low-density lipoprotein(OxLDL)-induced stress and inflammation in human endothelial cells and its underlying mechanisms.Methods:HUVECs were treated with OxLDL and/or fucoxanthin for a range of time points and concentrations.We evaluated the effects of fucoxanthin on OxLDL-induced HUVECs using the MTT assay,reactive oxygen species accumulation assay,ELISA,RT-PCR,immunofluorescence,and Western blotting.Results:Fucoxanthin enhanced the cell viability in a dose dependent manner after OxLDL exposure.Furthermore,fucoxanthin pretreatment significantly decreased OxLDL-induced reactive oxygen species production and prevented the activation of the nuclear factor kappa-B pathway,which led to substantial suppression of pro-inflammatory gene expressions.OxLDL-induced upregulation of interleukin-6,intercellular adhesion molecule-1,vascular cell adhesion molecule-1,interleukin-1β,monocyte chemotactic protein-1,cyclooxygenase-1,and tumor necrosis factor-αwas significantly reduced by fucoxanthin.Conclusions:Fucoxanthin can inhibit OxLDL-induced vascular inflammation and oxidative stress in HUVECs by targeting Nrf2 signaling pathways.

Attenuation of lipopolysaccharide-induced neuroinflammatory events in BV-2 microglial cells by Moringa oleifera leaf extract

Objective: To determine the anti-neuroinflammatory activity of Moringa oleifera leaf extract(MLE) under lipopolysaccharide stimulation of mouse murine microglia BV2 cells in vitro. Methods: The cytotoxicity effect of MLE was investigated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide assay. The inflammatory response of BV-2 cells were induced with lipopolysaccharide. The generation of nitric oxide levels was determined by using Griess assay and the level of pro-inflammatory cytokines(IL-1β, IL-6 and TNF-α) was evaluated by ELISA kit. The expression of iNOS, COX-2 as well as IκB-ααwas carried out by immunoblot analysis. Results: MLE reduced the nitric oxide production in concentration-dependent manner, and maintained the viability of BV-2 microglial cells which indicated absence of toxicity. In addition, MLE repressed the activation of nuclear factor kappa B by arresting the deterioration of IκB-α, consequently resulted in suppression of cytokines expression such as COX-2 and iNOS. Conclusions: MLE inhibitory activities are associated with the inhibition of nuclear factor kappa B transcriptional activity in BV2 microglial cells. Thus MLE may offer a substantial treatment for neuroinflammatory diseases.

健脾化浊调脂方调控NOX/ROS/NF-κB通路对动脉粥样硬化大耳兔的抗氧化应激损伤作用研究

目的研究健脾化浊调脂方(Jianpi Huazhuo Tiaozhi Recipe,JHTR)对动脉粥样硬化(atherosclerosis,AS)大耳兔氧化应激损伤的影响,探讨JHTR抗AS的可能机制。方法60只大耳兔随机分为空白对照组、模型对照组、JHTR(低、中、高)剂量组、阳性对照组各10只。除空白对照组外,其余5组均喂以高脂饲料制备AS兔模型。JHTR低、中、高剂量组分别予1,2,4g/(kg·d)JHTR灌胃,阳性对照组予阿托伐他汀2mg/(kg·d)灌胃。药物干预10周后,ELISA法测定血清氧化低密度脂蛋白(ox-LDL)、丙二醛(MDA)、超氧化物歧化酶(SOD)含量,化学荧光法测定胸主动脉ROS含量,Western blot法和qPCR法分别检测胸主动脉NOX4、p22phox蛋白表达和IKK-α、IKK-β、NF-κB mRNA表达。结果JHTR能够降低ox-LDL、MDA含量,提高SOD活性,并且下调NOX/ROS/NF-κB通路中NOX4、p22phox的蛋白表达,ROS含量,IKK-α、IKK-β、NF-κB mRNA表达。结论JHTR对AS大耳兔起抗氧化应激损伤的作用,这种保护作用可能与其抑制NOX/ROS/NF-κB通路有关。

健脾化浊方通过NOX4/ROS/NF-κB通路调节动脉粥样硬化大鼠主动脉炎症及氧化应激反应的研究

目的:研究健脾化浊方通过NAPDH氧化酶4(NOX4)/活性氧簇(ROS)/核因子-κB(NF-κB)通路调节动脉粥样硬化(AS)大鼠主动脉炎症及氧化应激反应的作用。方法:SD大鼠随机分为正常对照组、模型对照组、健脾化浊方10 g/kg组、20 g/kg组、40 g/kg组和健脾化浊方40 g/kg+内毒素(LPS)150μg/kg组,建立AS模型后灌胃给予不同剂量健脾化浊方或联合NF-κB激动剂LPS腹腔注射。检测主动脉粥样硬化的病理改变、气滞血瘀标志物及主动脉中炎症细胞因子、氧化应激指标、NOX4/ROS/NF-κB通路分子。结果:与模型对照组比较,健脾化浊方10 g/kg组、20 g/kg组、40 g/kg组大鼠的低切血粘度、中切血粘度、高切血粘度、血清中ET-1、GMP-140的含量及主动脉中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、细胞间黏附分子-1(ICAM-1)、C反应蛋白(CRP)、丙二醛(MDA)、晚期氧化蛋白产物(AOPP)、ROS的含量明显降低、NOX4、NF-κB的蛋白表达均明显下调,血清中一氧化氮(NO)、降钙素基因相关肽(CGRP)的含量及主动脉中总抗氧化力(T-AOC)的含量均明显升高;与健脾化浊方40 g/kg组比较,健脾化浊方40 g/kg+LPS 150μg/kg组大鼠的低切血粘度、中切血粘度、高切血粘度、血清中ET-1、GMP-140的含量及主动脉中TNF-α、IL-1β、ICAM-1、CRP、MDA、AOPP、ROS的含量明显增加、NOX4、NF-κB的蛋白表达量均明显上调,血清中NO、CGRP的含量及主动脉中T-AOC的含量明显降低。结论:健脾化浊方能够改善AS大鼠主动脉的AS病理改变及气滞血瘀表现,该作用与抑制NOX4/ROS/NF-κB通路介导的炎症反应及氧化应激反应有关。

Synergistic effects of particulate matter(PM_10) and SO_2 on human non-small cell lung cancer A549 via ROS-mediated NF-κB activation

Since a real atmospheric scenario usually represents a system involving multiple pollutants, air pollution studies typically focused on describing adverse effects associated with exposure to individual pollutants cannot reflect actual health risk. Particulate matter（PM10） and sulfur dioxide（SO2） are two major pollutants derived from coal combustion processes and co-existing in coal-smoke air pollution, but their potentially synergistic toxicity remains elusive thus far. In this study, we investigated the cytotoxic responses of PM10 and SO2, singly and in binary mixtures, using human non-small cell lung cancer A549 cells, followed by clarifying the possible mechanisms for their interaction. The results indicated that the concomitant treatment of PM10 and SO2 at low concentrations led to synergistic injury in terms of cell survival and apoptosis occurrence, while PM10 and SO2 alone at the same concentrations did not cause damage to the cells. Also, radical oxygen species（ROS） production followed by nuclear factor kappa B（NF-κB） activation was involved in the above synergistic cytotoxicity, which was confirmed by the repression of the actions by an ROS inhibitor（NAC）. This implies that assessment of health risk should consider the interactions between ambient PM and gaseous copollutants.

Osthole ameliorates myonecrosis caused by Clostridium perfringens type A infection in mice

This study aimed to investigate the protective effect of the nature product osthole(OST)against Clostridium perfrin-gens type A infection-caused myonecrosis in a mouse model.Male mice were divided into(1)control,(2)infected,(3)OST50 and(4)OST100 treatment groups.In the infected groups,mice were intramuscularly injected with 1×10^(8) CFU of C.perfringens per day for 6 days.Mice in the OST50 and OST100 groups were administrated intraperitoneally with OST at the doses of 50 or 100 mg/kg per day post C.perfringens infection.Our results showed that C.perfringens infection caused marked necrosis and inflammatory cell infiltration in the muscle tissues of mice.Mice in the OST50 and OST100 treatment groups displayed significantly attenuated C.perfringens infection-induced lipid peroxida-tion,oxidative stress,and apoptosis in their muscle tissue.Furthermore,OST treatment significantly downregulated the expressions of NF-κB,IL-1β,and TNF-αmRNA and protein levels,while concomitantly upregulating the expressions of Nrf2 and HO-1 mRNA and protein.OST treatments also inhibited the expression of phosphorylation(p)-p38,p-mTOR,and p-Erk1/2 proteins,and upregulated LC3II and Beclin1 proteins.In summary,our results reveal that OST therapy confers a protective effect against C.perfringens infection-induced oxidative stress and inflammation in muscle tissue,via activation of Nrf2/HO-1 and autophagy pathways and inhibition of p38,Erk1/2 and NF-κB pathways.

Photothermal therapy with regulated Nrf2/NF-κB signaling pathway for treating bacteria-induced periodontitis

Periodontitis is an inflammatory disease initiated by bacterial infection,developed by excessive immune response,and aggravated by high level of reactive oxygen species(ROS).Hence,herein,a versatile metal-organic framework(MOF)-based nanoplatform is prepared using mesoporous Prussian blue(MPB)nanoparticles to load BA,denoted as MPB-BA.The established MPB-BA nanoplatform serves as a shelter and reservoir for vulnerable immunomodulatory drug BA,which possesses antioxidant,anti-inflammatory and anti-bacterial effects.Thus,MPB-BA can exert its antioxidant,anti-inflammatory functions through scavenging intracellular ROS to switch macrophages from M1 to M2 phenotype so as to relieve inflammation.The underlying molecular mechanism lies in the upregulation of phosphorylated nuclear factor erythroid 2-related factor 2(Nrf2)to scavenge ROS and subsequently inhibit the nuclear factor kappa-B(NF-κB)signal pathway.Moreover,MPB-BA also exhibited efficient photothermal antibacterial activity against periodontal pathogens under near-infrared(NIR)light irradiation.In vivo RNA sequencing results revealed the high involvement of both antioxidant and anti-inflammatory pathways after MPB-BA application.Meanwhile,micro-CT and immunohistochemical staining of p-Nrf2 and p-P65 further confirmed the superior therapeutic effects of MPB-BA than minocycline hydrochloride.This work may provide an insight into the treatment of periodontitis by regulating Nrf2/NF-κB signaling pathway through photothermal bioplatform-assisted immunotherapy.

Toll-like receptor 2-mediated NF-kappa B pathway activation in ocular surface epithelial cells

Background:Gram-positive bacteria stimulate Toll-like receptor(TLR)2 and then activate the pro-inflammatory nuclear factor-kappa B(NF-κB)pathway.As the human ocular surface is heavily colonised by gram-positive cocci bacteria,a balance of activation/repression of NF-κB target genes is essential to avoid uncontrolled infection or autoimmune-related inflammation.It is advantageous to test NF-κB targeting molecules in an ocular surface culture system that allows assessment of temporal NF-κB activation in a longitudinal fashion without destruction of cells.Such initial testing under standardised conditions should reduce the number of molecules that progress to further evaluation in animal models.This study aims to establish an in-vitro cell culture system to assess NF-κB activation in the context of ocular surface cells.Methods:NF-κB activity was evaluated through a secretory alkaline phosphatase reporter assay(SEAP).Immunoblots and immunofluorescence were used to examine IκBαphosphorylation and p65/p50 nuclear localization.Monocyte chemoattractant protein-1(MCP-1)transcripts were evaluated by real time PCR and protein levels were measured by ELISA.Results:NF-κB activity in HCE-T cells treated with TLR2 activator Pam3CSK4 was higher than control cells at both 6 and 24 h.Pam3CSK4-stimulated NF-κB activation was inhibited by IκK inhibitors,Wedelolactone and BMS-345541.In Pam3CSK4 treated cells,active NF-κB subunits p50 and p65 increased in cell nuclear fractions as early as 1.5 h.Although the level of total IκB-αremained constant,phospho-IκB-αincreased with treatment over time.In the culture media of Pam3CSK4-stimulated cells,MCP-1 protein level was increased,which was suppressed in the presence of IκK inhibitors.Conclusion:NF-κB pathway can be activated by the TLR2 ligand and inhibited by IκK inhibitors in the ocular surface cell culture system.This cell culture system may be used to evaluate TLR-related innate defences in ocular surface diseases.

Discovery of novel aporphine alkaloid derivative as potent TLR2 antagonist reversing macrophage polarization and neutrophil infiltration against acute inflammation

Toll-like receptor 2(TLR2)mediated macrophages regulate the protective immune response to infectious microorganisms,but the aberrant activation of macrophages often leads to pathological inflammation,including tissue damage.In this study,we identified antagonists of TLR2 by screening2100 natural products and subsequently identified Taspine,an aporphine alkaloid,as an excellent candidate.Furthermore,analysis of the 10 steps chemical synthesis route and structural optimization yielded the Taspine derivative SMU-Y6,which has higher activity,better solubility,and improved drug-feasible property.Mechanistic studies and seq-RNA analysis revealed that SMU-Y6 inhibited TLR2 over other TLRs,hindered the formation of TLR2/MyD88 complex,and blocked the downstream NF-κB and MAPK signaling pathway,thus suppressing the release of inflammatory cytokines.SMU-Y6 could stabilize TLR2 and bind to TLR2 protein with a Kdof 0.18μmol/L.Additionally,SMU-Y6 could efficiently reverse the M1 phenotype macrophage polarization,reduce the production of cytokines as well as infiltration of neutrophiles and alleviate the local inflammation in mice with acute paw edema and colitis.Collectively,we reported the first aporphine alkaloid derivative that selectively inhibits TLR2 with high binding affinity and superior drug-feasible property,thus providing an urgently-needed molecular probe and potential drug candidate for inflammatory and autoimmune disease therapy.