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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-879026

ABSTRACT

The concentrations of seven anti-inflammatory components in blood and tissues were determined by UPLC-MS/MS after oral administration of Tetrastigma hemsleyanum aerial part(THAA) in healthy and inflammatory pathological model rats. The determination was carried out by using positive and negative ion switching technique, and multiple reaction monitoring(MRM) mode. The tissue distributions of the seven components in different physiological states were compared, and the patterns and characteristics of the effective components of THAA were studied. The results revealed that the seven effective components have large drug-time-curve areas(AUC) in heart, brain, small intestine, and stomach in both normal rats and inflammatory pathological model rats. This suggests that the anti-inflammatory effective component groups in THAA extract can all penetrate the blood-brain barrier, and have a large distribution area in gastrointestinal tract. It is inferred that gastrointestinal reabsorption may be one of the causes of the bimodal distribution of the drug-time curve of the drug blood distribution graph. As compared to normal rats, the effective component groups in THAA extract have higher drug-time curve area(AUC) in heart, brain, small intestine, stomach, liver, spleen, lung, kidney, and muscle of inflammatory pathological model rats. Among them, the effective component groups have the largest distribution area in heart, brain, small intestine, and stomach. This suggests that the binding force of organ tissues and drugs in the body may change under pathological conditions. It is speculated that the heart, brain, small intestine, and stomach may be the target tissues of THAA to produce anti-inflammatory effect. The retention times of THAA effective component groups in various organ tissues of rats in different physiological states are all relatively short, and do not have much difference. This suggests that no effective component accumulates in body, and that the pathological state of inflammation does not affect the onset times of the effective component groups. This experiment elucidates the patterns and characteristics of the in vivo target-effecting tissue distribution of THAA anti-inflammatory extract, and provides an experimental basis for clinical treatment.


Subject(s)
Animals , Anti-Inflammatory Agents , Chromatography, Liquid , Plant Components, Aerial , Plant Extracts , Rats , Tandem Mass Spectrometry , Tissue Distribution
5.
Chinese Pharmacological Bulletin ; (12): 706-712, 2018.
Article in Chinese | WPRIM | ID: wpr-705112

ABSTRACT

Aim To prepare hyaluronic acid nanoparti-cles(Ade/GA-HA) using glycyrrhetinic acid modified hyaluronic acid as the carrier and adenine as a model drug, and analyze their physicochemical property and proliferation effect on Bel-7402 cells. Methods Gly-cyrrhetinic acid and hyaluronic acid were combined by chemical cross-linking method, dialysis and freeze-dr-ying,based on which Ade/GA-HA was prepared using ultrasonic method, and the particle size and Zeta po-tential were determined by Malvern laser particle analy-zer,and the morphology was observed by transmission electron microscopy, and the absorbance was deter-mined by ultraviolet-visible spectrophotometer, high performance liquid chromatograph and microplate read-er to caculate drug load, encapsulation efficiency and in vitro release. MTT assays were utilized to determine the proliferation of nanoparticles treated Bel-7402 cells. Results GA-HA nanoparticles had spherical shape with a good dispersion, at diameters of 398.1 nm, of which Zeta potential was - 34.2 mV, and presented good short term stability. The drug load and encapsulation efficiency of Ade/GA-HA nanoparticles were (22.5 ± 5.8)% and (87.27 ± 0.33) %, re-spectively. Burst release was observed in Ade/GA-HA nanoparticles within 4 h, while controlled release 4 h later. Compared with free adenine,Ade/GA-HA nano-particles had a stronger inhibitory effect on cell prolif-eration with statistically significant difference. Conclu-sion GA-HA nanoparticles has excellent physico-chemical properties and meet the design requirement.

6.
Chinese Pharmaceutical Journal ; (24): 999-1003, 2014.
Article in Chinese | WPRIM | ID: wpr-859714

ABSTRACT

OBJECTIVE: To develop a kind of Agkistrodon acutus (Günther) DNA test kit, evaluate its quality indexes including specificity, stability, sensitivity and repeatability, and inspect the qualities of commercialAgkistrodon acutus (Günther) samples. METHODS: The Agkistrodon acutus (Günther) DNA test kit was developed and modified according to the method recorded in Chinese Pharmacopoeia (2010 edition). EighteenAgkistrodon acutus (Günther) samples were randomly collected from Beijing, Tianjin, and Changchun. The kit assay was performed to identify these samples with the pharmacopoeia method as the standard control. RESULTS: The kit proved effective after 20 times of freezing and thawing, and repeatability test showed same data for three tests. The lower limit of quantitation was 0.025 g. The specificity test confirmed that 14 samples were genuine, and 4 were adulterants. All of the identification results by the kit assay were in accordance with the ones by the pharmacopoeia method. CONCLUSION: The developed DNA test kit is accurate and effective for identification of Agkistrodon acutus (Günther). Compared with the pharmacopoeia method, it is simpler and more rapid, demonstrating broad prospect in quality inspection of Agkistrodon acutus (Günther).

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