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1.
Article in Chinese | WPRIM | ID: wpr-928090

ABSTRACT

This study investigated the mechanism of baicalin on lipopolysaccharide(LPS)/interferon γ(IFN-γ)-induced inflammatory microglia based on the triggering receptor expressed on myeloid cells 2(TREM2)/Toll-like receptor 4(TLR4)/nuclear factor kappaB(NF-κB) pathway. Specifically, LPS and IFN-γ were used to induce inflammation in mouse microglia BV2 cells. Then the normal group, model group, low-dose(5 μmol·L~(-1)) baicalin group, medium-dose(10 μmol·L~(-1)) baicalin group, high-dose(20 μmol·L~(-1)) baicalin group, and minocycline(10 μmol·L~(-1)) group were designed. Cell viability was detected by CCK-8 assay and cell morphology was observed under bright field. The expression of interleukin-1β(IL-1β), interleukin-4(IL-4), inducible nitric oxide synthase(iNOS), interleukin-6(IL-6), interleukin-10(IL-10), and arginase-1(Arg-1) mRNA was detected by real-time quantitative PCR, the protein expression of tumor necrosis factor-α(TNF-α), IL-1β, TREM2, TLR4, inhibitor kappaB-alpha(IκBα), p-IκBα, NF-κB p65 and p-NF-κB p65 by Western blot, and transfer of NF-κB p65 from cytoplasm to nucleus by cellular immunofluorescence. Compared with the normal group, most of the BV2 cells in the model group tended to demonstrate the pro-inflammatory M1 amoeba morphology, and the model group showed significant increase in the mRNA levels of IL-1β, IL-6, and iNOS, decrease in the mRNA levels of IL-4, IL-10, and Arg-1(P<0.01), rise of the protein expression of TNF-α, IL-1β, TLR4, p-IκBα, and p-NF-κB p65(P<0.01), reduction in TREM2 protein expression, and increase in the expression of NF-κB p65 in nucleus. Compared with the model group, baicalin groups and minocycline group showed the recovery of BV2 cell morphology, significant decrease in the mRNA levels of IL-1β, IL-6 and iNOS, increase in the mRNA levels of IL-4, IL-10, and Arg-1(P<0.01), reduction in the protein expression of TNF-α, IL-1β, TLR4, p-IκBα, and p-NF-κB p65(P<0.05), rise of TREM2 protein expression, and decrease in the expression of NF-κB p65 in nucleus. In summary, these results suggest that baicalin can regulate the imbalance between TREM2 and TLR4 of microglia and inhibit the activation of downstream NF-κB, thus promoting the polarization of microglia from pro-inflammatory phenotype to anti-inflammatory phenotype.


Subject(s)
Animals , Flavonoids , Inflammation/genetics , Interferon-gamma , Lipopolysaccharides/adverse effects , Mice , NF-kappa B/metabolism , Toll-Like Receptor 4/metabolism
2.
Article in Chinese | WPRIM | ID: wpr-906265

ABSTRACT

Objective:To investigate the neuroprotective effects of Danggui Shaoyaosan (DSS) on APP<sub>swe</sub>/PS1<sub>ΔE9 </sub>transgenic (APP/PS1) mice and its mechanism related to circular RNA (circRNA). Method:Totally twenty 6-month-old APP/PS1 mice were divided into model group and DSS group, and 10 C57BL/6 wild-type mice were set as the normal control group. The normal group and model group received the same volume of normal saline, and DSS group received drug by gavage administration, all for 8 weeks. The differentially expressed circRNA of APP/PS1 mice before and after DSS intervention was analyzed by circRNA sequencing to construct circRNA-miRNA mRNA interaction network. The results of cricRNA sequencing were then verified by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR). The protein expression levels of phosphoinositide 3-kinase (PI3K), p-PI3K, protein kinase B1 (Akt1), p-Akt1, B lymphocytoma-2 (Bcl-2), and Bcl-2-Associated X protein (Bax) in the hippocampus were detected by immunoblotting (Western blot). The protein expression of Caspase-3 in the hippocampus was detected by immunohistochemistry and the level of apoptosis in the hippocampus was detected by the TUNEL method. Result:Compared with the model group, there were 90 differentially expressed circRNA after intervention with DSS, of which 46 were up-regulated and 44 down-regulated. Compared with the normal group, the expression levels of circRNA1398 and circRNA1399 in the model group decreased, and the expression levels of miR-103-3p, miR-153-3p, miR-143-3p, and miR-143-5p increased. Compared with the model group, the expression levels of circRNA1398 and circRNA1399 in the DSS group were up-regulated, while the expression levels of miR-103-3p, miR-153-3p, miR-143-3p, and miR-143-5p were down-regulated. Compared with the normal group, the expression of p-PI3K, Akt1, p-Akt1, and Bcl-2 in the model group decreased (<italic>P</italic><0.05,<italic> P</italic><0.01), and the expression of Bax and Caspase in the model group increased (<italic>P</italic><0.01). Compared with the model group, the expression of p-PI3K, Akt1, p-Akt1, and Bcl-2 in the hippocampus of the DSS group increased (<italic>P</italic><0.01), and the protein expression of Bax and Caspase decreased (<italic>P</italic><0.01). Compared with the normal group, the apoptosis level in the hippocampus of the model group increased, with an apoptosis rate of (43.76±2.92)%. Compared with the model group, the apoptosis rate of DSS group was (24.64±3.39)%. Conclusion:DSS can activate PI3K/Akt pathway and inhibit apoptosis in hippocampal neurons of APP / PS1 mice, and play a neuroprotective role. The specific mechanism may be related to the regulation of circRNA1398 and circRNA1399 expression and the corresponding miRNA expression.

3.
Article in Chinese | WPRIM | ID: wpr-906044

ABSTRACT

Objective:To investigate the neuroprotective effect of Danggui Shaoyaosan (DSS) in a rat model of amyloid-<italic>β</italic>-peptide<sub>1-42</sub> (A<italic>β</italic><sub>1-42</sub>)-induced Alzheimer's disease (AD) as well as its regulatory effect on NOD-like receptor protein 3 (NLRP3)/cysteinyl aspartate-specific protease-1 (Caspase-1) signaling pathway. Method:The AD animal model was established via intracerebral injection of A<italic>β</italic><sub>1-42</sub> and treated with different concentrations of DSS after the division of rats into the sham operation group, model group, as well as the high-, medium-, and low-dose DSS groups. Morris water maze test was conducted to determine the learning and memory abilities of rats. The morphology and function of neurons were detected by hematoxylin-eosin (HE) staining and Golgi staining, followed by immunofluorescence co-localization of NLRP3 inflammasome activation. The mRNA expression levels of interleukin (IL)-1<italic>β</italic> and IL-18 were measured by Real-time polymerase chain reaction (Real-time PCR), and the protein expression levels of NLRP3, Caspase-1, and IL-1<italic>β </italic>were assayed by Western blot. Result:Compared with the sham operation group, the model group exhibited significantly decreased learning and memory abilities (<italic>P</italic><0.01), impaired neuronal morphology and function, up-regulated IL-1<italic>β</italic> and IL-18 mRNA expression, enhanced NLRP3 inflammasome activation, and elevated NLRP3, Caspase-1, and IL-1<italic>β</italic> protein expression (<italic>P</italic><0.01). Compared with the model group, DSS at both medium and high doses remarkably improved the learning and memory abilities of AD rats (<italic>P</italic><0.05, <italic>P</italic><0.01), restored neuronal morphology and function, down-regulated the mRNA expression levels of inflammatory factors IL-1<italic>β</italic> and IL-18, reduced the activation of NLRP3 inflammasomes, and lowered the protein expression levels of NLRP3, Caspase-1, and IL-1<italic>β</italic> (<italic>P</italic><0.01). Conclusion:DSS inhibits inflammasome activation and neuroinflammatory response possibly by regulating the NLRP3/Caspase-1 signaling pathway, thus exerting the neuroprotective effect.

4.
Article in Chinese | WPRIM | ID: wpr-873241

ABSTRACT

Objective::To investigate the regulatory effect of Danggui Shaoyaosan (DSS)-containing serum on oxidative stress and inflammation in H2O2-induced SH-SY5Y cells. Method::Methyl thiazolyl tetrazolium(MTT) assay was used to determine the cell activity and construct the H2O2-induced cell damage model with the optimal time and dose. Normal group, model group and high, medium and low-dose DSS groups(2.5%, 5%, 10%) were set up. MTT method was used to detect cell activity, spectrophotometry anti-oxidation indexes of malonaldehyde (MDA), catalase (CAT) and superoxide dismutase (SOD) and glutathione (GSH). Real-time quantitative PCR (Real-time PCR) was used to detect tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) mRNA expressions. And immunofluorescence test was adopted to detect nuclear transcription factor-κB(NF-κB) p65 nuclear translocation of the DSS after the intervention. Result::After 24 h intervention with 250 μmol·L-1 H2O2, SH-SY5Y cell viability was about 55%, which was the best modeling condition. After high, medium and low-dose DSS intervention on H2O2-damaged cell model, compared with the model group, the cell activity showed a dose-dependent increase (P<0.05), MDA was significantly reduced (P<0.05), and antioxidant indexes CAT, SOD and GSH were significantly increased (P<0.05). H2O2 could significantly increase SH-SY5Y cell inflammatory factor TNF-α, IL-6 and IL-1β mRNA expressions, and promote activation of cytoplasmic NF-κB and nuclear translocation. DSS-containing serum showed a dose-dependent inhibition of NF-κB p65 from nuclear, and reduced inflammatory factor levels, such as TNF-α, IL-6 and IL-1β. Conclusion::DSS-containing serum can significantly reduce the oxidative damage in H2O2-induced SH-SY5Y cells by improving their antioxidant status, and reduce the inflammatory response by inhibiting NF-κB signaling pathway.

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