Protective Mechanism of Banxia Houputang on Lipopolysaccharide-induced Neuroinflammatory Injury / 中国实验方剂学杂志

ObjectiveTo study the inhibitory effect of Banxia Houputang （BHT） on lipopolysaccharide （LPS）-induced inflammation of microglia （BV2） cells and the neuroprotective effect on human neuroblastoma （SH-SY5Y） cells. MethodAfter the neuroinflammatory model was constructed by LPS inducing BV2 cells， model group （LPS 100 µg·L-1）， administration groups （LPS+1 g·L-1 BHT， LPS+2 g·L-1 BHT， LPS+5 g·L-1 BHT， LPS+10 g·L-1 BHT）， and blank group were given DEME medium at the same volume. In addition， neuronal apoptosis model was established by co-culture of LPS-induced BV2 cell inflammation medium and SH-SY5Y cells （LPS-DMEM） and was administrated according to the above grouping. Cell viability was detected by Cell Counting Kit-8 （CCK-8） assay. The content of nitric oxide （NO） and that of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， and interleukin-6 （IL-6） were determined by Griess aasay and enzyme-linked immunosorbent assay （ELISA）， respectively. The mRNA levels of TNF-α， IL-1β， interleukin-4 （IL-4）， nitric oxide synthase （iNOS）， and interleukin-10 （IL-10） were measured by real-time polymerase chain reaction （Real-rime PCR）. Western blot was used to detect the expression levels of signal transducer and activator of transcription 3 （STAT3）， Janus kinase 2 （JAK2） and nuclear factor kappa-B （NF-κB p65）， protein kinase B （Akt）， inhibitor of nuclear factor κB α （IκBα）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2 associated X protein （Bax）. ResultCompared with blank group， LPS increased the NO release， levels of TNF-α， IL-1β， IL-6， and iNOS and protein expression of Akt， NF-κB p65， IκBα， JAK2 and STAT3， decreased the content of IL-4 and IL-10 in BV2 cells， and induced apoptosis of co-cultured SH-SY5Y cells （P<0.01）. Compared with model group， BHT reduced the content of NO， TNF-α， IL-1β， and iNOS （P<0.01） and protein expression of Akt， NF-κB p65， IκBα， JAK2 and STAT3 （P<0.01）， elevated the content of IL-4 and IL-10 （P<0.01）， and inhibited the apoptosis of SH-SY5Y cells induced by LPS-DMEM （P<0.01）. ConclusionThis experiment reveals that BHT inhibited LPS-induced inflammation in BV2 cells by regulating Akt/NF-κB/JAK2/STAT3 signaling pathway and showed neuroprotective effects on SH-SY5Y cells.

Two new polyacetylene glycosides from rhizomes of Atractylodes lancea / 中草药

Objective: To investigate the chemical components from the 80% EtOH extract of Atractylodes lancea, as well as the inhibitory activities of the isolated compounds on LPS-induced NO production of microglia BV2 cells. Methods: The n-BuOH-soluble fraction of the crude extract was successively chromatographed with Diaion HP-20, Sephadex LH-20, and preparative HPLC C18-column. At last, the planar and stereochemical structures of these obtained compounds were established on the basis of extensive spectroscopic data (HRESIMS, NMR, and ECD, etc). Results: Ten glycosides were isolated from the n-BuOH-soluble fraction of the 80% EtOH extract of A. lancea, including (2E,8R)-decene-4,6-diyne-1,8-diol-1-O-β-D- apiofuranosyl-(1→6)-β-D-glucopyranoside (1), (8S)-decane-4,6-diyne-1,8-diol-8-O-β-D-glucopyranoside (2), (2E,8R)-decene-4,6- diyne-1,8-diol-8-O-β-D-glucopyranoside (3), (2E,8S)-decene-4,6-diyne-1,8-diol-8-O-β-D-glucopyranoside (4), (2E,8E)-2,8- decadiene-4,6-diyne-1,10-diol-1-O-β-D-glucopyranoside (5), (7R,8S)-3',9,9'-trihydroxyl-3-methoxyl-1'-propanol-7,8-dihydrobenzo- funanneoligan-4-O-β-D-glucopyranoside (6), (7'R*,8S*,8'S*)-lyoniresinol 9'-O-β-D-glucopyranoside (7), (7S,8R)-4,9,9'-trihydroxy- 3'-methoxy-8-O-4'-neolignan 7-O-β-D-glucopyranoside (8), methyl salicylate 2-O-α-L-xylopyranosyl-(1→6)-β-D-glucopyranoside (9), and phenylmethanol 7-O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (10). Conclusion: Compounds 1 and 2 are named atractyeneyneglycoside A and atractyeneyneglycoside B, while compounds 6, 8-10 are first isolated from the rhizomes of A. lancea. At the concentration of 10 μmol/L, compound 10 exhibited the strongest inhibitory effects on LPS-induced NO production of microglia BV2 cells with the value of 31.18%, while compounds 1 and 2 just showed weaker inhibitory effects with values of 22.01% and 14.09%, respectively.

Induction of Cell Death by Betulinic Acid through Induction of Apoptosis and Inhibition of Autophagic Flux in Microglia BV-2 Cells

Betulinic acid (BA), a natural pentacyclic triterpene found in many medicinal plants is known to have various biological activity including tumor suppression and anti-inflammatory effects. In this study, the cell-death induction effect of BA was investigated in BV-2 microglia cells. BA was cytotoxic to BV-2 cells with IC50 of approximately 2.0 μM. Treatment of BA resulted in a dose-dependent chromosomal DNA degradation, suggesting that these cells underwent apoptosis. Flow cytometric analysis further confirmed that BA-treated BV-2 cells showed hypodiploid DNA content. BA treatment triggered apoptosis by decreasing Bcl-2 levels, activation of capase-3 protease and cleavage of PARP. In addition, BA treatment induced the accumulation of p62 and the increase in conversion of LC3-I to LC3-II, which are important autophagic flux monitoring markers. The increase in LC3-II indicates that BA treatment induced autophagosome formation, however, accumulation of p62 represents that the downstream autophagy pathway is blocked. It is demonstrated that BA induced cell death of BV-2 cells by inducing apoptosis and inhibiting autophagic flux. These data may provide important new information towards understanding the mechanisms by which BA induce cell death in microglia BV-2 cells.

Inhibitory effect of Corilagin on the inflammatory response of irradiated microglia BV-2 cells / 中华放射医学与防护杂志

Objective To explore the inhibitory effects of Corilagin on the production of proinflammatory cytokines in microglia induced by radiation. Methods The cytotoxicity of Corilagin was measured by MTT assay. Microglia BV-2 cells were irradiated 0 or 32 Gy after pretreated with Corilagin for 12 hours. Realtime-PCR was used to detect the mRNA levels of inflammatory cytokines, such as IL-1β,TNF-α on several time-points. The content of nitric oxide (NO) was determined with nitrate reductase method. The translocation of NF-κB was measured by Western blot and immunocytochemical stain.Confocal microscopy was used to observe the expression of Iba-1 and Nemo. Results No cytotoxicity was detected on BV-2 cells with 1-10 μg/ml Corilagin. Iba-1 expression in microglia cells was activated by irradiation, the expression levels of inflammatory cytokines, such as IL-1β, TNF-α and NO were also elevated. Whereas, the production of IL-1 β, TNF-α in activated microglia cells was significantly inhibited with 5 μg/mL corilagin ( tIL-1β = 6. 341, tTNF-α = 3.41 1, tNO = 3. 134, P ＜ 0. 05 ). Corilagin significantly inhibited the expression of Nemo and the translocation of NF-κB p65. Conclusion Corilagin could inhibit the activation of irradiated microglia cells and down-regulate the expression of inflammatory cytokines, via inhibition of the NF-κB signaling pathway.

PrP 106-126 Altered PrP mRNA Gene Expression in Mouse Microglia BV-2 Cells' / 中国病毒学·英文版

Prion diseases are infectious and fatal neurodegenerative diseases. The pathogenic agent is an abnormal prion protein aggregate. Microglial activation in the centre nervous system is a characteristic feature of prion disease. In this study, we examined the effect of PrP 106-126 on PrP mRNA gene expression in Mouse microglia cells BV-2 by real-time quantitative PCR. PrP mRNA expression level was found to be significantly increased after 18 h exposure of BV-2 cells to PrP 106-126, with 3-fold increase after 18 h and 4.5-fold increase after 24 h and BV-2 cells proliferating occurred correspondingly. Our results provide the first in vitro evidence of the increase of PrP mRNA levels in microglial cells exposed to PrP 106-126, and indicate that microglial cells might play a critical role in prion pathogenesis.