Baicalin inhibits streptozotocin-induced neuroinflammation in Alzheimer' s disease rat model by TLR4/MyD88/NF-κB pathway北大核心CSCD

Aim To investigate the effects of baicalin on the inflammatory response and Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD 88)/nuclear factor kappa B (N F-K B) signaling pathway in Alzheimer' s disease (AD) rat model induced by lateral ventricular injection of streptozotocin (STZ). Methods The AD animal model was constructed by lateral ventricular injection of STZ in SD rats, and divided into sham operation group, model group, low-dose (60 mg -1 • kg-1 • d-1 ) baicalin group, high-dose (120 mg-1 • kg-1 • d-1 ) baicalin group and minocycline group (36 mg-1 • kg-1 • d-1 ). The learning and memory ability of rats were assessed by Morris water maze. The mRNA expressions of tumor necrosis Factor-α(TNF-α), interleukin-6 (IL-6), interleukin-1β(IL-1β) were detected by real-time quantitative PCR, the protein expressions of TLR4, MyD88, N F-Κ B p65, p-NF-κB p65 and nucleus/cytoplasm NF-ΚB p65 were detected by Western blot, and NF-ΚB p65 transfering from cytoplasm to nucleus and the activation of microglia were measured by immunofluorescence. Results Compared with the sham operation group, animals in the model group showed decreased learning and memory capacity, increased mRNA expression of TNF-α, IL-6 and IL-1κ, up-regulated protein expressions of TLR4 and MyD88, the ratio of p-NF-κB p65/NF-κB p65 and the nucleus/cytoplasm ratio of NF-ΚB p65, enhanced expression of NF-KB p65 in nucleus and the activation of microglias. Compared with the model group, baicalin group and minocycline group improved the learning and memory capacity of AD rats, decreased the mRNA expression of TNF-α, IL-6 and IL-1β, down-regulated the protein expressions of TLR4 and MyD88, the p-NF-ΚB P65/NF-KB p65 ratio and the nucleus/cytoplasm ratio of NF-KB p65, and reduced the expression of NF-KB p65 in nucleus and the activation of microglias. Conclusion Baicalin may exert neuroprotective effects by inhibiting the TLR4/MyD88/NF-KB signaling pathway and neuroinflammation response in the AD brain. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

Inhibitory effect of 1,8-cineol （eucalyptol） on Egr-1 expression in lipopolysaccharide-stimulated THP-1 cells

Aim： To study the effects of 1,8-cineol （eucalyptol） on the expression of early growth response factor-1 （Egr-1） and NF-κB in the human monocyte THP-1 cell line stimulated by lipopolysaccharide （LPS）. Methods： The THP-1 cells were incu- bated with serial doses of 1,8-cineol （1, 10, and 100 mg/L, 30 min） before being stimulated with LPS （1 mg/L, 30 min）. The localization of Egr-1 in the THP-1 cells was detected by immunofluorescence and a laser scanning confocal microscope. The expression of Egr-1 in the nuclei and whole cell, and NF-κB in the nuclei, were measured by Western blot analysis. Results： When stimulated by LPS, the FITC- labeled Egr-1 was detected mainly in the nuclei. Moreover, the expression of Egr-1 in the whole cell increased markedly compared with the control cells. 1,8- Cineol pretreatment decreased the expression of Egr-1 in both the nuclei and whole cell of the LPS-stimulated THP- 1 cells, and this effect was concentration- dependent, but there was no reaction on the expression of NF-κB in the nuclei protein in the LPS-stimulated THP-1 cells. Conclusion： In a concentration-depen- dent manner, 1,8-Cineol reduces LPS-induced Egr-1 expression in nuclei and in whole cell of THP-1 cells, but shows no effect on NF-κB expression.     

Inhibitory effect of compound W3D on LPS-induced release of inflammatory mediators of RAW264.7 cells through TLR4-MyD88-NF-kB signaling pathway北大核心CSCD

Aim To investigate the effect of the novel benzoxazolone derivative 4-(5'-dimethylamino)-naph-thalenesulfonyl-2 (3 H)-benzoxazolone (W3D) on TLR4-MyD88-NF-KB signaling pathway in LPS-induced RAW264. 7 cells. Methods The cell viability was detected by MTT assay, and the contents of TNF-a, IL-6, IL-lβ and COX-2 in the cell supernatant were analyzed using ELISA methods. The protein expression of IL-6, TLR4, MyD88, p-IRAK4 and NF-kB were investigated by western blot analysis, and the mRNA expressions of TLR4, MyD88 and IL-6 were analyzed by RT-PCR. Results W3D could obviously inhibit the production of TNF-ct, IL-6 and IL-1 β in LPSinduced RAW264.7 cell supernatant, but it had no effect on the release of COX-2. Compared with the model group, the expressions of TLR4, MyD88 and IL-6 were decreased significantly in a dose dependent manner. Meanwhile, the expressions of p-IRAK4 and nucleus of NF-kB were decrease in W3D treated group compared with the model group. Conclusion The novel compound W3D could inhibit the release of the inflammatory mediators through the regulation of TLR4-MyD88-NF-rcB signaling pathway. © 2018 Publication Centre of Anhui Medical University. All rights reserved.     

Effect of α-pinene on nuclear translocation of NF-κB in THP-1 cells

AIM: To study the effects of α-pinene on nuclear translocation of nuclear factor-κB (NF-κB) and the expression of the inhibitor of NF-κB (IκBα) in human monocyte THP-1 cell line. METHODS: THP-1 cells were incubated with α-pinene (1, 10, and 100 mg/L, for 30 min) before being stimulated with lipopolysaccharide (LPS, 1 mg/L, 30 min).The location of NF-κB p65 subunit (NF-κB/p65) in THP-1 cells was detected by immunofluorescence and laser scanning confocal microscope (LSCM). The expression of NF-κB/p65 in nuclei and that of IκBα in cytoplasm were measured by Western-blot analysis. RESULTS: The majority of FITC-labelled NF-κB/p65 was located in the nuclei being stimulated with LPS. Whereas, no such fluorescence was seen in the nuclei of the groups pretreated with α-pinene or control cells, α-Pinene pretreatment decreased the NF-κB/p65 nuclear translocation in LPS-stimulated THP-1 cells, and this effect was dose-dependent, but there was no reaction in LPS-unstimulated THP-1 cells, α-Pinene pretreatment increased IκBα protein level in cytoplasm, compared with that in LPS-stimulated THP-1 cells. CONCLUSION: In a dose-related fashion, α-pinene inhibits the nuclear translocation of NF-κB induced by LPS in THP- 1 cells, and this effect is partly due to the upregulation of IκBα expression.     

Compound FLZ inhibits lipopolysaccharide-induced inflammatory effects via down-regulation of the TAK-IKK and TAK-JNK/ p38MAPK pathways in RAW264.7 macrophages

Aim： The aim of this study was to investigate the effect of the squamosamide derivative FLZ （N-2-（4-hydroxy-phenyl）-ethyl-2-（Z,5-dimethoxy-phenyl）-3-（3-methoxy-4-hydroxy-phenyl）-acrylamide） on lipopolysaccharide （LPS）-induced inflam-matory mediator production and the underlying mechanism in RAW264.7 macrophages. Methods： RAW264.7 cells were preincubated with non-toxic concentrations of compound FLZ （1, 5, and 10 μmol/L） for 30 min and then stimulated with 10 μg/L LPS. The production of nitric oxide （NO）, the expression of inducible nitric oxide synthase （iNOS） and cyclooxygenase 2 （COX-2）, and the activation of nuclear factor kappa-B （NF-KB） and mitogen-activated protein kinase （MAPK） pathways were examined. Results： FLZ significantly inhibited the LPS-induced production of NO, as well as the expression of iNOS and COX-2 at both the RNA and the protein levels in RAW264.7 cells. The LPS-induced increase in the DNA binding activity of NF-KB and activator protein i （AP-1）, the nuclear translocation of NF-κB p65, the degradation of the inhibitory κBα protein （IκBα） and the phosphorylation of IκBα, IκB kinase （IKK） α/β, c-Jun NH2-terminal kinase （JNK） and p38 MAPKs were all suppressed by FLZ. However, the phosphorylation of extracellular signal-regulated kinase （ERK） was not affected. Further study revealed that FLZ inhibited the phosphorylation of transforming growth factor-β （TGF-β）-activated kinase 1（TAK1）, which is an upstream signaling molecule required for IKKα/β, JNK and p38 activation. Conclusion： FLZ inhibited the LPS-induced production of inflammatory mediators at least partly through the downregulation of the TAK-IKK and TAK-JNK/p38MAPK pathways.     

The artemisinin analog SM934 alleviates dry eye disease in rodent models by regulating TLR4/NF-κB/NLRP3 signaling

Dry eye disease(DED)is a multifactorial disorder of the tears and ocular surface characterized by manifestations of dryness and irritation.Although the pathogenesis is not fully illuminated,it is recognized that inflammation has a prominent role in the development and deterioration of DED.β-aminoarteether maleate(SM934)is a water-soluble artemisinin derivative with antiinflammatory and immunosuppressive activities.In this study,we established scopolamine hydrobromide(SCOP)-induced rodent model as well as benzalkonium chloride(BAC)-induced rat model to investigate the therapeutic potential of SM934 for DED.We showed that topical application of SM934(0.1%,0.5%)significantly increased tear secretion,maintained the number of conjunctival goblet cells,reduced corneal damage,and decreased the levels of inflammatory mediators(TNF-α,IL-6,IL-10,or IL-1β)in conjunctiva in SCOP-induced and BAC-induced DED models.Moreover,SM934 treatment reduced the accumulation of TLR4-expressing macrophages in conjunctiva,and suppressed the expression of inflammasome components,i.e.,myeloid differentiation factor88(MyD88),Nod-like receptor protein 3(NLRP3),apoptosis-associated speck-like protein containing CARD(ASC),and cleaved caspase 1.In LPS-treated RAW 264.7 cells,we demonstrated that pretreatment with SM934(10μM)impeded the upregulation of TLR4 and downstream NF-κB/NLRP3 signaling proteins.Collectively,artemisinin analog SM934 exerts therapeutic benefits on DED by simultaneously reserving the structural integrity of ocular surface and preventing the corneal and conjunctival inflammation,suggested a further application of SM934 in ophthalmic therapy,especially for DED.     

The protective effect of L-Shikonin on LPS/D-GalN-induced acute liver injury in mice via inhibiting NF-κB inflammatory signaling pathway北大核心CSCD

Aim To investigate the anti-inflammatory effect of L-Shikonin ( SK ) on lipopolysaccharide ( LPS)-induced RAW 264. 7 macrophages in vitro and its protective effect on LPS/D-GalN-induced acute liver injury. Methods The mouse model of acute liver in¬jury was established in vivo experiments by LPS/D- GalN. The survival rate of the mice and the changes of liver and spleen indices in each group were examined. The levels of AST, ALT and AKP in serum and NO, superoxide dismutase ( SOD ) and malondialdehyde (MDA) in liver tissue homogenate were measured, and the histopathological sections of the liver of each group were observed by H&E staining. M I T colorimet- ric assay was used for cell viability in vitro experi¬ments, Griess method for the detection of NO content, RT-PCR assay and Western blot assay for examining the effect of levulinic acid on the expression levels of mRNA and related pathway proteins of pro-inflammato¬ry factors in LPS-induced RAW264. 7 cells. Results The results of in vivo experiments showed that L-SK significantly improved the liver and spleen indices, de¬creased AST, ALT and AKP levels in serum, de¬creased NO and MDA in liver homogenate, and in¬creased SOD activity in mice with acute liver injury. The results of in vitro experiments showed that L-SK significantly inhibited the mRNA expression of INOS, COX2, I FN-(3 and pro-inflammatory factors 1L-6, TNF-a and IL-10 in LPS-induced RAW264. 7 cells, and significantly inhibited the protein expression of IN¬OS, COX2 and the NF-kB signaling pathway. Conclu¬sions L-SK has good anti-inflammatory effects in LPS-induced inflammation in RAW 264. 7 cells in vitro. Il inhibits the protein expression of phosphorylated P65 and IKKaαβ in the NF-kB signaling pathway, thereby suppressing the anti-inflammatory effects in vitro and L- Shikonin has protective effects against acute liver injury in mice. © 2023 Publication Centre of Anhui Medical University. All rights reserved.     

石蒜碱通过TLR4/NF-κB途径抑制LPS诱导的原代小胶质细胞炎症反应

目的 观察石蒜碱（LYC）对脂多糖（LPS）诱导的原代小胶质细胞炎症反应和表型变化的影响,并探讨其作用机制。方法 将培养的原代小胶质细胞分为对照组、LYC组（0.1、0.5、1和5 μmol/L LYC）、LPS组（0.1 mg/L LPS）和LPS（0.1 mg/L LPS）+LYC组（5 μmol/L LYC）。倒置显微镜观察细胞形态变化;流式细胞术检测原代小胶质细胞的纯度及LPS诱导原代小胶质细胞向两种表型转化的情况;CCK-8法检测细胞活性;实时荧光定量PCR（qPCR）检测白细胞介素（IL）-1β、IL-6、肿瘤坏死因子-α（TNF-α）以及不同表型小胶质细胞表面标志物mRNA表达;Griess法检测一氧化氮（NO）含量;免疫印迹法检测细胞TLR4、p-NF-κB p65蛋白表达情况。结果 原代小胶质细胞纯度达95%以上。对照组及各浓度LYC组细胞活性差异无统计学意义。后续实验以5 μmol/L的LYC为药物浓度。与LPS组相比,LPS+LYC组的阿米巴样小胶质细胞数量明显减少,IL-1β、IL-6、TNF-α mRNA表达水平以及NO含量降低,CD86 mRNA表达水平和表型比例降低,CD206 mRNA表达水平和表型比例升高,TLR4和p-NF-κB p65蛋白表达水平降低（P<0.01）。结论 LYC通过TLR4/NF-κB信号通路抑制LPS诱导的原代小胶质细胞炎症反应,并促进其向“抑炎型”极化。     

9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol, from Eulophia ochreata, inhibits inflammatory signalling mediated by Toll-like receptors

Background and purpose:

9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol (RSCL-0520) is a phenanthrene isolated from Eulophia ochreata, one of the Orchidaceae family, known by local tradition to exhibit medicinal properties. However, no anti-inflammatory activity or any molecular mechanisms involved have been reported or elucidated. Here, for the first time, we evaluate the anti-inflammatory properties of RSCL-0520 on responses induced by lipopolysaccharide (LPS) and mediated via Toll-like receptors (TLRs).

Experimental approach:

The in vitro anti-inflammatory activities of RSCL-0520 were investigated in LPS-stimulated monocytic cells, measuring activation of cytokine and inflammatory genes regulated by nuclear factor-κB (NF-κB). Tumour necrosis factor (TNF)-α levels in serum following LPS stimulation in mice and carrageenan-induced paw oedema in rats were used as in vivo models.

Key results:

Pretreatment with RSCL-0520 effectively inhibited LPS-induced, TLR4-mediated, NF-κB-activated inflammatory genes in vitro, and reduced both LPS-induced TNF-α release and carrageenan-induced paw oedema in rats. Treatment with RSCL-0520 reduced LPS-stimulated mRNA expression of TNF-α, COX-2, intercellular adhesion molecule-1, interleukin (IL)-8 and IL-1β, all regulated through NF-κB activation. RSCL-0520, however, did not interfere with any cellular processes in the absence of LPS.

Conclusions and implications:

RSCL-0520 blocked signals generated by TLR4 activation, as shown by down-regulation of NF-κB-regulated inflammatory cytokines. The inhibitory effect involved both MyD88-dependent and -independent signalling cascades. Our data elucidated the molecular mechanisms involved, and support the search for plant-derived TLR antagonists, as potential anti inflammatory agents.     

Plumbagin inhibits cell growth and potentiates apoptosis in human gastric cancer cells in vitro through the NF-κB signaling pathway

Aim:

To investigate the effects and underlying mechanisms of plumbagin, a naphthoquinone derived from medicinal plant Plumbago zeylanica, on human gastric cancer (GC) cells.

Methods:

Human gastric cancer cell lines SGC-7901, MKN-28, and AGS were used. The cell viability was examined using CCK-8 viability assay. Cell proliferation rate was determined using both clonogenic assay and EdU incorporation assay. Apoptosis was detected via Annexin V/propidium iodide double-labeled flow cytometry. Western blotting was used to assess the expression of both NF-κB-regulated gene products and TNF-α-induced activation of p65, IκBα, and IKK. The intracellular location of NF-κB p65 was detected using confocal microscopy.

Results:

Plumbagin (2.5–40 μmol/L) concentration-dependently reduced the viability of the GC cells. The IC₅₀ value of plumbagin in SGC-7901, MKN-28, and AGS cells was 19.12, 13.64, and 10.12 μmol/L, respectively. The compound (5–20 μmol/L) concentration-dependently induced apoptosis of SGC-7901 cells, and potentiated the sensitivity of SGC-7901 cells to chemotherapeutic agents TNF-αand cisplatin. The compound (10 μmol/L) downregulated the expression of NF-κB-regulated gene products, including IAP1, XIAP, Bcl-2, Bcl-xL, tumor factor (TF), and VEGF. In addition to inhibition of NF-κB p65 nuclear translocation, the compound also suppressed TNF-α-induced phosphorylation of p65 and IKK, and the degradation of IκBα.

Conclusion:

Plumbagin inhibits cell growth and potentiates apoptosis in human GC cells through the NF-κB pathway.